Use of ezh2 inhibitors for treating cancer

ABSTRACT

The disclosure provides methods of treating, identifying and/or selecting for treatment a subject having a cancer in which an immune checkpoint protein is upregulated. In certain embodiments, the methods for treating cancer in a subject in need thereof comprise administering to the subject: (a) a therapeutically effective amount of an EZH2 inhibitor and (b) a therapeutically effective amount of an immune checkpoint inhibitor. In certain embodiments of the methods of the disclosure, the EZH2 inhibitor is tazemetostat.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/618,518, filed Dec. 2, 2019, which is a U.S. National Phaseapplication, filed under 35 U.S.C. § 371, of International ApplicationNo. PCT/US2018/035655, filed Jun. 1, 2018, which claims priority to, andthe benefit of, U.S. Provisional Application Nos. 61/514,535, filed Jun.2, 2017, and 62/530,814, filed Jul. 10, 2017, the contents of each ofwhich are incorporated herein by reference in their entireties.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled“EPIZ-082_C01US_SeqList.xml” created on Jan. 31, 2023, which is 4,289bytes in size. The information in the electronic format of the sequencelisting is incorporated herein by reference in its entirety.

SUMMARY

Some aspects of the present disclosure provide strategies and methodsuseful for enhancing the treatment of certain cancers and for theablation of hyperproliferative cells in vivo, ex vivo, and in vitro.Some aspects of this disclosure are based on the recognition thattreatment of cancer with certain anti-cancer drugs can result in changesin target cells (e.g., hyperproliferative cells) or target tissues(e.g., tumor tissue), that enhance survival and/or proliferation of thetarget cells or tissues, thus counteracting the effect of theanti-cancer therapy.

For example, some aspects of the present disclosure are based on therecognition that certain anti-cancer drugs increase the number orpercentage of cells in a tumor that express genes improving survivaland/or proliferation in such cells, including, for example, genes thatmodulate immune evasion of the target cells. Some aspects of thisdisclosure are based on the recognition that genes modulating immuneevasion of tumor cells are expressed or upregulated in certain cancersafter treatment with certain anti-cancer drugs.

Some aspects of the present disclosure provide methods for identifying asubject having a cancer expressing an immune checkpoint protein aftertreatment with an anti-cancer drug; methods for selecting a subject fortreatment, e.g., for treatment with a combination of an anti-cancer drugand an immune checkpoint inhibitor, based on the subject having a cancerin which an immune checkpoint protein is expressed or upregulated; andmethods of treating a subject having a cancer expressing an immunecheckpoint protein after treatment with an anti-cancer drug, e.g., byadministering a combination of an anti-cancer drug and an immunecheckpoint inhibitor to the subject.

The present disclosure also provides methods comprising detectingexpression of an immune checkpoint protein in a subject. Some aspects ofthe present disclosure are based on the recognition that certain immunecheckpoint proteins (e.g. PD-L1) are expressed or upregulated in somesubjects upon treatment with certain anti-cancer drugs, e.g., aftertreatment with EZH2 inhibitors. Further aspects of the presentdisclosure are based on the recognition that combining treatmentmodalities or strategies employing an epigenetic modulator, e.g., anEZH2 inhibitor, with those employing an immune checkpoint inhibitorresults in an advantageous combination therapy approach for treatingcertain diseases, e.g., certain proliferative diseases.

Some aspects of the disclosure relate to methods comprising detectingexpression of an immune checkpoint protein in a subject having cancer.In some embodiments, the subject has been administered an effectiveamount of an enhancer of zeste homolog 2 (EZH2) inhibitor. Suitableimmune checkpoint proteins and methods for their detection are disclosedherein, and additional suitable immune checkpoint proteins and detectionmethods will be apparent to those of average skill in the art based onthe present disclosure. The disclosure is not limited in this aspect.

Some aspects of the disclosure relate to methods comprisingadministering to a subject having a cancer expressing an immunecheckpoint protein an effective amount of an EZH2 inhibitor and aneffective amount of an immune checkpoint inhibitor.

In some embodiments, the methods of the disclosure comprise detecting alevel of expression of an immune checkpoint protein in the subject afterthe subject has been administered an EZH2 inhibitor (e.g., a first EZH2inhibitor), and comparing the level of expression to a reference level.In some embodiments, the reference level is a level of immune checkpointprotein expression observed in the subject before administration of theEZH2 inhibitor.

In some embodiments, the methods of the disclosure further compriseadministering an immune checkpoint inhibitor to the subject, if (a)immune checkpoint protein expression is detected in the subject, or (b)if the level of an immune checkpoint protein expression detected in thesubject after administration of an EZH2 inhibitor (e.g., a first EZH2inhibitor) is higher than the reference level.

In some embodiments, the methods of the disclosure further compriseselecting the subject as a candidate for a treatment with an immunecheckpoint inhibitor if the presence of an immune checkpoint protein inthe subject is detected.

In some embodiments, the methods of the disclosure further compriseadministering an effective amount of an EZH2 inhibitor (e.g., a secondEZH2 inhibitor) to the subject. In further embodiments, methods of thedisclosure comprise administering the EZH2 inhibitor after the detectionof an immune checkpoint protein expression in the subject.

In some embodiments, the EZH2 inhibitor that has been administered tothe subject prior to detection of expression of an immune checkpointprotein (e.g., the first EZH2 inhibitor) and the EZH2 inhibitor that isbeing administered to a subject that is selected for treatment (e.g.,the second EZH2 inhibitor) are the same. In some embodiments, the EZH2inhibitor that has been administered to the subject prior to detectionof expression of an immune checkpoint protein (e.g., the first EZH2inhibitor) and the EZH2 inhibitor that is being administered to asubject that is selected for treatment (e.g., the second EZH2 inhibitor)are different from each other.

In some embodiments, the expression of an immune checkpoint protein inthe cancer of the subject has been detected after administration of aneffective amount of an EZH2 inhibitor. In even further embodiments, thesubject has received an EZH2 inhibitor (e.g., the first EZH2 inhibitor),and the expression of the immune checkpoint protein in the cancer washigher after the administration of the EZH2 inhibitor than before theadministration of the EZH2 inhibitor.

In some embodiments, the methods of the disclosure comprise detecting aT-cell marker in the cancer of the subject. In further embodiments, thesubject has a cancer that is positive for a T-cell marker. In evenfurther embodiments, the subject has a cancer that positive for a T-cellmarker after administration of an EZH2 inhibitor (e.g., the first EZH2inhibitor). In some embodiments, the T-cell marker comprises CD4. Insome embodiments, the T-cell marker comprises CD8. Suitable T-cellmarkers and methods for their detection are disclosed herein, andadditional suitable T-cell markers and detection methods will beapparent to those of skill in the art based on the present disclosure.The disclosure is not limited in this aspect.

In some embodiments of the disclosure, the immune checkpoint protein isprogrammed death-ligand 1 (PD-L1).

Some aspects of the disclosure provide methods for treating cancer in asubject having cancer in which an immune checkpoint protein is expressedor upregulated, the methods comprising administering to the subject: (a)a therapeutically effective amount of an EZH2 inhibitor and (b) atherapeutically effective amount of an immune checkpoint inhibitor.

Some aspects of the disclosure relate to an EZH2 inhibitor in atherapeutically effective amount for use in the treatment of cancer in asubject having cancer in which an immune checkpoint protein is expressedor upregulated by co-administration with an immune checkpoint inhibitorin a therapeutically effective amount.

Some aspects of the disclosure relate to an EZH2 inhibitor in atherapeutically effective amount for use as a medicament for thetreatment of cancer in a subject having cancer in which an immunecheckpoint protein is expressed or upregulated by co-administration withan immune checkpoint inhibitor in a therapeutically effective amount.

Some aspects of the disclosure relate to the use of an EZH2 inhibitor ina therapeutically effective amount in the manufacture of a medicamentfor the treatment of cancer in a subject having cancer in which animmune checkpoint protein is expressed or upregulated byco-administration with an immune checkpoint inhibitor in atherapeutically effective amount.

Some aspects of the disclosure relate to an EZH2 inhibitor in atherapeutically effective amount for use in combination with an immunecheckpoint inhibitor in a therapeutically effective amount in thetreatment of cancer in a subject having cancer in which an immunecheckpoint protein is expressed or upregulated.

Some aspects of the disclosure relate to the use of an EZH2 inhibitor ina therapeutically effective amount as a medicament for combinationaltherapy with an immune checkpoint inhibitor in a therapeuticallyeffective amount in the treatment of cancer in a subject having cancerin which an immune checkpoint protein is expressed or upregulated.

Some aspects of the disclosure relate to the use of an EZH2 inhibitor ina therapeutically effective amount in a combinational therapy with animmune checkpoint inhibitor in a therapeutically effective amount in thetreatment of cancer in a subject having cancer in which an immunecheckpoint protein is expressed or upregulated.

Some aspects of the disclosure relate to the use of an EZH2 inhibitor ina therapeutically effective amount in the manufacture of a medicamentfor combinational therapy with an immune checkpoint inhibitor in atherapeutically effective amount in the treatment of cancer in a subjecthaving cancer in which an immune checkpoint protein is expressed orupregulated.

Some aspects of the disclosure relate to an immune checkpoint inhibitorin a therapeutically effective amount for use in the treatment of cancerin a subject having cancer in which an immune checkpoint protein isexpressed or upregulated by co-administration with an EZH2 inhibitor ina therapeutically effective amount.

Some aspects of the disclosure relate to an immune checkpoint inhibitorin a therapeutically effective amount for use as a medicament for thetreatment of cancer in a subject having cancer in which an immunecheckpoint protein is expressed or upregulated by co-administration withan EZH2 inhibitor in a therapeutically effective amount.

Some aspects of the disclosure relate to the use of an immune checkpointinhibitor in a therapeutically effective amount in the manufacture of amedicament for the treatment of cancer in a subject having cancer inwhich an immune checkpoint protein is expressed or upregulated byco-administration with an EZH2 inhibitor in a therapeutically effectiveamount.

Some aspects of the disclosure relate to an immune checkpoint inhibitorin a therapeutically effective amount for use in combination with anEZH2 inhibitor in a therapeutically effective amount in the treatment ofcancer in a subject having cancer in which an immune checkpoint proteinis expressed or upregulated.

Some aspects of the disclosure relate to the use of an immune checkpointinhibitor in a therapeutically effective amount as a medicament forcombinational therapy with an EZH2 inhibitor in a therapeuticallyeffective amount in the treatment of cancer in a subject having cancerin which an immune checkpoint protein is expressed or upregulated.

Some aspects of the disclosure relate to the use of an immune checkpointinhibitor in a therapeutically effective amount in a combinationaltherapy with an EZH2 inhibitor in a therapeutically effective amount inthe treatment of cancer in a subject having cancer in which an immunecheckpoint protein is expressed or upregulated.

Some aspects of the disclosure relate to the use of an immune checkpointinhibitor in a therapeutically effective amount in the manufacture of amedicament for combinational therapy with an EZH2 inhibitor in atherapeutically effective amount in the treatment of cancer in a subjecthaving cancer in which an immune checkpoint protein is expressed orupregulated.

In certain embodiments, the subject has a cancer expressing PD-L1. Incertain embodiments, the expression of PD-L1 in the cancer of thesubject has been detected after administration of an effective amount ofan EZH2 inhibitor (e.g., the first EZH2 inhibitor). In certainembodiments, the subject has received an EZH2 inhibitor (e.g., the firstEZH2 inhibitor), and wherein the expression of PD-L1 in the cancer ishigher after the administration of the EZH2 inhibitor than before theadministration of the EZH2 inhibitor.

Some aspects of the present disclosure provide methods comprisingadministering an EZH2 inhibitor to a subject who is already receiving animmune checkpoint inhibitor, e.g., as part of an ongoing treatmentstrategy for a proliferative disease that the subject has been diagnosedwith. In some embodiments, the EZH2 inhibitor is administered to thesubject already receiving the immune checkpoint inhibitor based on therecognition that the proliferative disease in the subject is sensitiveto treatment with the EZH2 inhibitor, or to a combination of the EZH2inhibitor and the immune checkpoint inhibitor.

Some aspects of the present disclosure provide methods comprisingadministering an immune checkpoint inhibitor to a subject who is alreadyreceiving an EZH2 inhibitor, e.g., as part of an ongoing treatmentstrategy for a proliferative disease that the subject has been diagnosedwith. In some embodiments, the immune checkpoint inhibitor isadministered to the subject already receiving the EZH2 inhibitor basedon the recognition that the proliferative disease in the subject issensitive to treatment with the immune checkpoint inhibitor, or to acombination of the EZH2 inhibitor and the immune checkpoint inhibitor.In certain embodiments, the subject has a cancer in which expression ofan immune checkpoint protein is upregulated after treatment with ananti-cancer drug, e.g., as compared to a reference level, or as comparedto the pre-treatment expression level. In certain embodiments, thesubject has a cancer expressing PD-L1. In certain embodiments, theexpression of PD-L1 in the cancer of the subject is detected after thesubject has been administered the EZH2 inhibitor (e.g., the first EZH2inhibitor). In certain embodiments, the expression of PD-L1 in thecancer is higher after the administration of the EZH2 inhibitor thanbefore the administration of the EZH2 inhibitor.

Some aspects of this disclosure provide methods of administering (a) atherapeutically effective amount of an EZH2 inhibitor and (b) atherapeutically effective amount of an immune checkpoint inhibitor to asubject in need thereof, e.g., to a subject having or diagnosed with aproliferative disease (e.g., a cancer in which an immune checkpointprotein, such as PD-L1, is upregulated, e.g., after the subject receivesan EZH2 inhibitor), based on the recognition that the disease in thesubject is sensitive to combined treatment with the EZH2 inhibitor andthe immune checkpoint inhibitor. In some embodiments, the disease in thesubject is not sensitive to treatment with the EZH2 inhibitor and/or theimmune checkpoint inhibitor alone.

Some aspects of this disclosure provide methods for treating a diseasein a subject, e.g., a proliferative disease, by administering (a) anEZH2 inhibitor and (b) an immune checkpoint inhibitor to the subject,wherein the disease cannot effectively be treated or a clinicallydesirable endpoint cannot be reached by administering the EZH2 inhibitoralone or by administering the immune checkpoint inhibitor alone. In someembodiments, the method comprises administering an EZH2 inhibitor and/oran immune checkpoint inhibitor to the subject at a dosage that is lowerthan the minimal effective dose for administering the EZH2 inhibitoralone or the minimal effective dose for administering the immunecheckpoint inhibitor alone.

Some aspects of the disclosure relate to an EZH2 inhibitor in atherapeutically effective amount for use in the treatment of a diseasein a subject, e.g., a proliferative disease, by co-administration withan immune checkpoint inhibitor in a therapeutically effective amount,wherein the disease cannot effectively be treated or a clinicallydesirable endpoint cannot be reached by administering the EZH2 inhibitoralone or by administering the immune checkpoint inhibitor alone.

Some aspects of the disclosure relate to an EZH2 inhibitor in atherapeutically effective amount for use as a medicament for thetreatment of a disease in a subject, e.g., a proliferative disease, byco-administration with an immune checkpoint inhibitor in atherapeutically effective amount, wherein the disease cannot effectivelybe treated or a clinically desirable endpoint cannot be reached byadministering the EZH2 inhibitor alone or by administering the immunecheckpoint inhibitor alone.

Some aspects of the disclosure relate to the use of an EZH2 inhibitor ina therapeutically effective amount in the manufacture of a medicamentfor the treatment of a disease in a subject, e.g., a proliferativedisease, by co-administration with an immune checkpoint inhibitor in atherapeutically effective amount, wherein the disease cannot effectivelybe treated or a clinically desirable endpoint cannot be reached byadministering the EZH2 inhibitor alone or by administering the immunecheckpoint inhibitor alone.

Some aspects of the disclosure relate to an EZH2 inhibitor in atherapeutically effective amount for use in combination with an immunecheckpoint inhibitor in a therapeutically effective amount in thetreatment of a disease in a subject, e.g., a proliferative disease,wherein the disease cannot effectively be treated or a clinicallydesirable endpoint cannot be reached by administering the EZH2 inhibitoralone or by administering the immune checkpoint inhibitor alone.

Some aspects of the disclosure relate to the use of an EZH2 inhibitor ina therapeutically effective amount as a medicament for combinationaltherapy with an immune checkpoint inhibitor in a therapeuticallyeffective amount in the treatment of a disease in a subject, e.g., aproliferative disease, wherein the disease cannot effectively be treatedor a clinically desirable endpoint cannot be reached by administeringthe EZH2 inhibitor alone or by administering the immune checkpointinhibitor alone.

Some aspects of the disclosure relate to the use of an EZH2 inhibitor ina therapeutically effective amount in a combinational therapy with animmune checkpoint inhibitor in a therapeutically effective amount in thetreatment of a disease in a subject, e.g., a proliferative disease,wherein the disease cannot effectively be treated or a clinicallydesirable endpoint cannot be reached by administering the EZH2 inhibitoralone or by administering the immune checkpoint inhibitor alone.

Some aspects of the disclosure relate to the use of an EZH2 inhibitor ina therapeutically effective amount in the manufacture of a medicamentfor combinational therapy with an immune checkpoint inhibitor in atherapeutically effective amount in the treatment of a disease in asubject, e.g., a proliferative disease, wherein the disease cannoteffectively be treated or a clinically desirable endpoint cannot bereached by administering the EZH2 inhibitor alone or by administeringthe immune checkpoint inhibitor alone.

Some aspects of the disclosure relate to an immune checkpoint inhibitorin a therapeutically effective amount for use in the treatment of adisease in a subject, e.g., a proliferative disease, byco-administration with an EZH2 inhibitor in a therapeutically effectiveamount, wherein the disease cannot effectively be treated or aclinically desirable endpoint cannot be reached by administering theEZH2 inhibitor alone or by administering the immune checkpoint inhibitoralone.

Some aspects of the disclosure relate to an immune checkpoint inhibitorin a therapeutically effective amount for use as a medicament for thetreatment of a disease in a subject, e.g., a proliferative disease, byco-administration with an EZH2 inhibitor in a therapeutically effectiveamount, wherein the disease cannot effectively be treated or aclinically desirable endpoint cannot be reached by administering theEZH2 inhibitor alone or by administering the immune checkpoint inhibitoralone.

Some aspects of the disclosure relate to the use of an immune checkpointinhibitor in a therapeutically effective amount in the manufacture of amedicament for the treatment of a disease in a subject, e.g., aproliferative disease, by co-administration with an EZH2 inhibitor in atherapeutically effective amount, wherein the disease cannot effectivelybe treated or a clinically desirable endpoint cannot be reached byadministering the EZH2 inhibitor alone or by administering the immunecheckpoint inhibitor alone.

Some aspects of the disclosure relate to an immune checkpoint inhibitorin a therapeutically effective amount for use in combination with anEZH2 inhibitor in a therapeutically effective amount in the treatment ofa disease in a subject, e.g., a proliferative disease, wherein thedisease cannot effectively be treated or a clinically desirable endpointcannot be reached by administering the EZH2 inhibitor alone or byadministering the immune checkpoint inhibitor alone.

Some aspects of the disclosure relate to the use of an immune checkpointinhibitor in a therapeutically effective amount as a medicament forcombinational therapy with an EZH2 inhibitor in a therapeuticallyeffective amount in the treatment of a disease in a subject, e.g., aproliferative disease, wherein the disease cannot effectively be treatedor a clinically desirable endpoint cannot be reached by administeringthe EZH2 inhibitor alone or by administering the immune checkpointinhibitor alone.

Some aspects of the disclosure relate to the use of an immune checkpointinhibitor in a therapeutically effective amount in a combinationaltherapy with an EZH2 inhibitor in a therapeutically effective amount inthe treatment of a disease in a subject, e.g., a proliferative disease,wherein the disease cannot effectively be treated or a clinicallydesirable endpoint cannot be reached by administering the EZH2 inhibitoralone or by administering the immune checkpoint inhibitor alone.

Some aspects of the disclosure relate to the use of an immune checkpointinhibitor in a therapeutically effective amount in the manufacture of amedicament for combinational therapy with an EZH2 inhibitor in atherapeutically effective amount in the treatment of a disease in asubject, e.g., a proliferative disease, wherein the disease cannoteffectively be treated or a clinically desirable endpoint cannot bereached by administering the EZH2 inhibitor alone or by administeringthe immune checkpoint inhibitor alone.

In some embodiments, administering both inhibitors at such sub-minimaldosages is useful to avoid side effects associated with administeringthe agents at higher dosages, while still achieving a clinical desirableoutcome.

Some aspects of the disclosure relate to a product comprising an EZH2inhibitor in a therapeutically effective amount and an immune checkpointinhibitor in a therapeutically effective amount as a combinedpreparation for simultaneous, separate or sequential use in thetreatment of a disease.

Some aspects of the disclosure relate to a kit comprising (a) apharmaceutical composition comprising an EZH2 inhibitor in atherapeutically effective amount and (b) a pharmaceutical compositioncomprising an immune checkpoint inhibitor in a therapeutically effectiveamount.

Some aspects of the disclosure relate to a synergistic composition of anEZH2 inhibitor in a therapeutically effective amount and an immunecheckpoint inhibitor in a therapeutically effective amount, wherein theEZH2 inhibitor and the immune checkpoint inhibitor come into contactwith each other in the body of the subject (e.g., only in the body ofthe subject).

Some aspects of the disclosure relate to a method of preparing asynergistic composition of an EZH2 inhibitor in a therapeuticallyeffective amount and an immune checkpoint inhibitor in a therapeuticallyeffective amount by bringing the EZH2 inhibitor and the immunecheckpoint inhibitor into contact with each other at a locus (e.g.,within the body of the subject).

Some aspects of the disclosure provide combinations and compositionscomprising (a) a therapeutically effective amount of an EZH2 inhibitorand (b) a therapeutically effective amount of an immune checkpointinhibitor.

In some embodiments, EZH2 inhibitors of the disclosure comprise acompound of Formula (Ig) or a pharmaceutically acceptable salt thereof:

wherein R₂, R₄ and R₁₂ are each, independently C₁₋₆ alkyl;

R₆ is C₆-C₁₀ aryl or 5- or 6-membered heteroaryl, each of which isoptionally substituted with one or more -Q₂-T₂, wherein Q₂ is a bond orC₁-C₃ alkyl linker optionally substituted with halo, cyano, hydroxyl orC₁-C₆ alkoxy, and T₂ is H, halo, cyano, —OR_(a), —NR_(a)R_(b),—(NR_(a)R_(b)R_(c))⁺A⁻, —C(O)R_(a), —C(O)OR_(a), —C(O)NR_(a)R_(b),—NR_(b)C(O)R_(a), —NR_(b)C(O)OR_(a), —S(O)₂R_(a), —S(O)₂NR_(a)R_(b), orR_(S2), in which each of R_(a), R_(b), and R_(c), independently is H orR_(S3), A⁻ is a pharmaceutically acceptable anion, each of R_(S2) andR_(S3), independently, is C₁-C₆ alkyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4to 12-membered heterocycloalkyl, or 5- or 6-membered heteroaryl, orR_(a) and R_(b), together with the N atom to which they are attached,form a 4 to 12-membered heterocycloalkyl ring having 0 or 1 additionalheteroatom, and each of R_(S2), R_(S3), and the 4 to 12-memberedheterocycloalkyl ring formed by R_(a) and R_(b), is optionallysubstituted with one or more -Q₃-T₃, wherein Q₃ is a bond or C₁-C₃ alkyllinker each optionally substituted with halo, cyano, hydroxyl or C₁-C₆alkoxy, and T₃ is selected from the group consisting of halo, cyano,C₁-C₆ alkyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-memberedheterocycloalkyl, 5- or 6-membered heteroaryl, OR_(d), COOR_(d),—S(O)₂R_(d), —NR_(d)R_(e), and —C(O)NR_(d)R_(e), each of R_(d) and R_(e)independently being H or C₁-C₆ alkyl, or -Q₃-T₃ is oxo; or any twoneighboring -Q₂-T₂, together with the atoms to which they are attachedform a 5- or 6-membered ring optionally containing 1-4 heteroatomsselected from N, O and S and optionally substituted with one or moresubstituents selected from the group consisting of halo, hydroxyl, COOH,C(O)O—C₁-C₆ alkyl, cyano, C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino,di-C₁-C₆ alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-memberedheterocycloalkyl, and 5- or 6-membered heteroaryl;

R₇ is -Q₄-T₄, in which Q₄ is a bond, C₁-C₄ alkyl linker, or C₂-C₄alkenyl linker, each linker optionally substituted with halo, cyano,hydroxyl or C₁-C₆ alkoxy, and T₄ is H, halo, cyano, NR_(f)R_(g),—OR_(f), —C(O)R_(f), —C(O)OR_(f), —C(O)NR_(f)R_(g), —C(O)NR_(f)OR_(g),—NR_(f)C(O)R_(g), —S(O)₂R_(f), or R_(S4), in which each of R_(f) andR_(g), independently is H or R_(S5), each of R_(S4) and R_(S5),independently is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, or 5- or6-membered heteroaryl, and each of R_(S4) and R_(S5) is optionallysubstituted with one or more -Q₅-T₅, wherein Q₅ is a bond, C(O),C(O)NR_(k), NR_(k)C(O), S(O)₂, or C₁-C₃ alkyl linker, R_(k) being H orC₁-C₆ alkyl, and T₅ is H, halo, C₁-C₆ alkyl, hydroxyl, cyano, C₁-C₆alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, 5- or6-membered heteroaryl, or S(O)_(q)R_(q) in which q is 0, 1, or 2 andR_(q) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl,C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, or 5- or 6-memberedheteroaryl, and T₅ is optionally substituted with one or moresubstituents selected from the group consisting of halo, C₁-C₆ alkyl,hydroxyl, cyano, C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-memberedheterocycloalkyl, and 5- or 6-membered heteroaryl except when T₅ is H,halo, hydroxyl, or cyano; or -Q₅-T₅ is oxo; and

R₈ is H, halo, hydroxyl, COOH, cyano, R_(S6), OR_(S6), or COOR_(S6), inwhich R_(S6) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈cycloalkyl, 4 to 12-membered heterocycloalkyl, amino, mono-C₁-C₆alkylamino, or di-C₁-C₆ alkylamino, and R_(S6) is optionally substitutedwith one or more substituents selected from the group consisting ofhalo, hydroxyl, COOH, C(O)O—C₁-C₆ alkyl, cyano, C₁-C₆ alkoxyl, amino,mono-C₁-C₆ alkylamino, and di-C₁-C₆ alkylamino; or R₇ and R₈, togetherwith the N atom to which they are attached, form a 4 to 11-memberedheterocycloalkyl ring having 0 to 2 additional heteroatoms, and the 4 to11-membered heterocycloalkyl ring formed by R₇ and R₈ is optionallysubstituted with one or more -Q₆-T₆, wherein Q₆ is a bond, C(O),C(O)NR_(m), NR_(m)C(O), S(O)₂, or C₁-C₃ alkyl linker, R_(m) being H orC₁-C₆ alkyl, and T₆ is H, halo, C₁-C₆ alkyl, hydroxyl, cyano, C₁-C₆alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, 5- or6-membered heteroaryl, or S(O)_(p)R_(p) in which p is 0, 1, or 2 andR_(p) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl,C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, or 5- or 6-memberedheteroaryl, and T₆ is optionally substituted with one or moresubstituents selected from the group consisting of halo, C₁-C₆ alkyl,hydroxyl, cyano, C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-memberedheterocycloalkyl, and 5- or 6-membered heteroaryl except when T₆ is H,halo, hydroxyl, or cyano; or -Q₆-T₆ is oxo.

In certain embodiments of Formula (Ig), R₆ is C₆-C₁₀ aryl or 5- or6-membered heteroaryl, each of which is optionally, independentlysubstituted with one or more -Q₂-T₂, wherein Q₂ is a bond or C₁-C₃ alkyllinker, and T₂ is H, halo, cyano, —OR_(a), —NR_(a)R_(b),—(NR_(a)R_(b)R_(c))⁺A⁻, —C(O)NR_(a)R_(b), —NR_(b)C(O)R_(a), —S(O)₂R_(a),or R_(S2), in which each of R_(a) and R_(b), independently is H orR_(S3), each of R_(S2) and R_(S3), independently, is C₁-C₆ alkyl, orR_(a) and R_(b), together with the N atom to which they are attached,form a 4 to 7-membered heterocycloalkyl ring having 0 or 1 additionalheteroatom, and each of R_(S2), R_(S3), and the 4 to 7-memberedheterocycloalkyl ring formed by R_(a) and R_(b), is optionally,independently substituted with one or more -Q₃-T₃, wherein Q₃ is a bondor C₁-C₃ alkyl linker and T₃ is selected from the group consisting ofhalo, C₁-C₆ alkyl, 4 to 7-membered heterocycloalkyl, OR_(a),—S(O)₂R_(a), and —NR_(d)R_(e), each of R_(d) and R_(e) independentlybeing H or C₁-C₆ alkyl, or -Q₃-T₃ is oxo; or any two neighboring -Q₂-T₂,together with the atoms to which they are attached form a 5- or6-membered ring optionally containing 1-4 heteroatoms selected from N, Oand S.

In some embodiments, EZH2 inhibitors of the disclosure comprise acompound of Formula (II) or a pharmaceutically acceptable salt thereof:

wherein Q₂ is a bond or methyl linker, T₂ is H, halo, —OR_(a),—NR_(a)R_(b), —(NR_(a)R_(b)R_(c))⁺A⁻, or —S(O)₂NR_(a)R_(b), R₇ ispiperidinyl, tetrahydropyran, cyclopentyl, or cyclohexyl, eachoptionally substituted with one -Q₅-T₅ and R₈ is ethyl.

In some embodiments, EZH2 inhibitors of the disclosure comprise acompound of Formula (IIa) or a pharmaceutically acceptable salt thereof:

wherein

each of R_(a) and R_(b), independently is H or R_(S3), R_(S3) beingC₁-C₆ alkyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-memberedheterocycloalkyl, or 5- or 6-membered heteroaryl, or R_(a) and R_(b),together with the N atom to which they are attached, form a 4 to12-membered heterocycloalkyl ring having 0 or 1 additional heteroatom,and each of R_(S3) and the 4 to 12-membered heterocycloalkyl ring formedby R_(a) and R_(b), is optionally substituted with one or more -Q₃-T₃,wherein Q₃ is a bond or C₁-C₃ alkyl linker each optionally substitutedwith halo, cyano, hydroxyl or C₁-C₆ alkoxy, and T₃ is selected from thegroup consisting of halo, cyano, C₁-C₆ alkyl, C₃-C₅ cycloalkyl, C₆-C₁₀aryl, 4 to 12-membered heterocycloalkyl, 5- or 6-membered heteroaryl,OR_(a), COOR_(d), —S(O)₂R_(a), —NR_(d)R_(e), and —C(O)NR_(d)R_(e), eachof R_(d) and R_(e) independently being H or C₁-C₆ alkyl, or -Q₃-T₃ isoxo;

R₇ is -Q₄-T₄, in which Q₄ is a bond, C₁-C₄ alkyl linker, or C₂-C₄alkenyl linker, each linker optionally substituted with halo, cyano,hydroxyl or C₁-C₆ alkoxy, and T₄ is H, halo, cyano, NR_(f)R_(g),—OR_(f), —C(O)R_(f), —C(O)OR_(f), —C(O)NR_(f)R_(g), —C(O)NR_(f)OR_(g),—NR_(f)C(O)R_(g), —S(O)₂R_(f), or R_(S4), in which each of R_(f) andR_(g), independently is H or R_(S5), each of R_(S4) and R_(S5),independently is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 7-membered heterocycloalkyl, or 5- or6-membered heteroaryl, and each of R_(S4) and R_(S5) is optionallysubstituted with one or more -Q₅-T₅, wherein Q₅ is a bond, C(O),C(O)NR_(k), NR_(k)C(O), S(O)₂, or C₁-C₃ alkyl linker, R_(k) being H orC₁-C₆ alkyl, and T₅ is H, halo, C₁-C₆ alkyl, hydroxyl, cyano, C₁-C₆alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 7-membered heterocycloalkyl, 5- or6-membered heteroaryl, or S(O)_(q)R_(q) in which q is 0, 1, or 2 andR_(q) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl,C₆-C₁₀ aryl, 4 to 7-membered heterocycloalkyl, or 5- or 6-memberedheteroaryl, and T₅ is optionally substituted with one or moresubstituents selected from the group consisting of halo, C₁-C₆ alkyl,hydroxyl, cyano, C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 7-memberedheterocycloalkyl, and 5- or 6-membered heteroaryl except when T₅ is H,halo, hydroxyl, or cyano; or -Q₅-T₅ is oxo; provided that R₇ is not H;and

R₈ is H, halo, hydroxyl, COOH, cyano, R_(S6), OR_(S6), or COOR_(S6), inwhich R_(S6) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, amino,mono-C₁-C₆ alkylamino, or di-C₁-C₆ alkylamino, and R_(S6) is optionallysubstituted with one or more substituents selected from the groupconsisting of halo, hydroxyl, COOH, C(O)O—C₁-C₆ alkyl, cyano, C₁-C₆alkoxyl, amino, mono-C₁-C₆ alkylamino, and di-C₁-C₆ alkylamino; or R₇and R₈, together with the N atom to which they are attached, form a 4 to11-membered heterocycloalkyl ring which has 0 to 2 additionalheteroatoms and is optionally substituted with one or more -Q₆-T₆,wherein Q₆ is a bond, C(O), C(O)NR_(m), NR_(m)C(O), S(O)₂, or C₁-C₃alkyl linker, R_(m) being H or C₁-C₆ alkyl, and T₆ is H, halo, C₁-C₆alkyl, hydroxyl, cyano, C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino,di-C₁-C₆ alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 7-memberedheterocycloalkyl, 5- or 6-membered heteroaryl, or S(O)_(p)R_(p) in whichp is 0, 1, or 2 and R_(p) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 7-membered heterocycloalkyl, or 5-or 6-membered heteroaryl, and T₆ is optionally substituted with one ormore substituents selected from the group consisting of halo, C₁-C₆alkyl, hydroxyl, cyano, C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino,di-C₁-C₆ alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 7-memberedheterocycloalkyl, and 5- or 6-membered heteroaryl except when T₆ is H,halo, hydroxyl, or cyano; or -Q₆-T₆ is oxo.

In certain embodiments of Formula (IIa), R_(a) and R_(b), together withthe N atom to which they are attached, form a 4 to 7-memberedheterocycloalkyl ring having 0 or 1 additional heteroatoms to the N atomand the ring is optionally substituted with one or more -Q₃-T₃, whereinthe heterocycloalkyl is azetidinyl, pyrrolidinyl, imidazolidinyl,pyrazolidinyl, oxazolidinyl, isoxazolidinyl, triazolidinyl, piperidinyl,1,2,3,6-tetrahydropyridinyl, piperazinyl, or morpholinyl.

In certain embodiments of Formula (IIa), R₇ is C₃-C₈ cycloalkyl or 4 to7-membered heterocycloalkyl, each optionally substituted with one ormore -Q₅-T₅.

In certain embodiments of Formula (IIa), R₇ is piperidinyl,tetrahydropyran, tetrahydro-2H-thiopyranyl, cyclopentyl, cyclohexyl,pyrrolidinyl, or cycloheptyl, each optionally substituted with one ormore -Q₅-T₅.

In certain embodiments of Formula (IIa), R₈ is H or C₁-C₆ alkyl which isoptionally substituted with one or more substituents selected from thegroup consisting of halo, hydroxyl, COOH, C(O)O—C₁-C₆ alkyl, cyano,C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, and di-C₁-C₆ alkylamino.

In some embodiments, EZH2 inhibitors of the disclosure comprise acompound is selected from

and a pharmaceutically acceptable salt thereof.

In some embodiments, EZH2 inhibitors of the disclosure comprise

or a pharmaceutically-acceptable salt thereof.

In some embodiments, the EZH2 inhibitor is tazemetostat. In someembodiments, the EZH2 inhibitor is the hydrobromide salt oftazemetostat. In some embodiments, the EZH2 inhibitor is thehydrochloride salt of tazemetostat.

In some embodiments, an immune checkpoint inhibitor of the disclosure isa CTLA4 inhibitor. In some embodiments, an immune checkpoint inhibitorof the disclosure targets, binds, or inhibits CTLA4. Exemplary suitableCTLA4 inhibitors of the disclosure include Ipilimumab, Ticilimumab,AGEN-1884 or a combination thereof.

In some embodiments, an immune checkpoint inhibitor of the disclosure isa PD-1 inhibitor. In some embodiments, an immune checkpoint inhibitor ofthe disclosure targets, binds, or inhibits PD-1 and/or PD-L1. Exemplarysuitable PD-1 and/or PD-L1 inhibitors include Nivolumab, Pembrolizumab,Atezolizumab, Durvalumab, Avelumab, BMS-936559, AMP-224, MEDI-0680,TSR-042, BGB-108, STI-1014, KY-1003, ALN-PDL, BGB-A317, KD-033,REGN-2810, PDR-001, SHR-1210, MGD-013, PF-06801591, CX-072 or acombination thereof. In certain embodiments, the immune checkpointinhibitor comprises Atezolizumab. In certain embodiments, the immunecheckpoint inhibitor comprises Nivolumab. In certain embodiments, theimmune checkpoint inhibitor comprises Pembrolizumab.

In some embodiments, an immune checkpoint inhibitor of the disclosure isan LAG3 inhibitor. In some embodiments, an immune checkpoint inhibitorof the disclosure targets, binds, or inhibits LAG3. Exemplary suitableLAG3 inhibitors include IMP-731, LAG-525, BMS-986016, GSK-2831781 or acombination thereof.

In some embodiments, an immune checkpoint inhibitor of the disclosure isan B7-H3 inhibitor. In some embodiments, an immune checkpoint inhibitorof the disclosure targets, binds, or inhibits B7-H3. Exemplary suitableB7-H3 inhibitors include Enoblituzumab, 1241-8H9, DS-5573 or acombination thereof.

In some embodiments, an immune checkpoint inhibitor of the disclosure isa Tim3 inhibitor. In some embodiments, an immune checkpoint inhibitor ofthe disclosure targets, binds, or inhibits Tim3. Exemplary suitable Tim3inhibitors include MBG-453.

Those of ordinary skill in the art will understand that the exemplaryimmune checkpoint inhibitors provided herein are non-limiting examples,and are not meant to limit the scope of the present disclosure.Additional suitable immune checkpoint inhibitors will be apparent to theskilled artisan based on the present disclosure and the generalknowledge in the art. The disclosure is not limited in this respect.

In some embodiments, the EZH2 inhibitor is a small molecule drug, e.g.,tazemetostat, or a pharmaceutically acceptable salt thereof. In someembodiments, the immune checkpoint inhibitor is a peptide, or protein,e.g., a monoclonal antibody, e.g., Nivolumab, Pembrolizumab,Atezolizumab, Durvalumab, or Avelumab. Based on the differentbioavailability and pharmacokinetics of small molecule drugs andtherapeutic peptides or proteins, the EZH2 inhibitor and the immunecheckpoint inhibitor are, in some embodiments, administered viadifferent routes and/or according to different administration schedules.

For example, in some embodiments, the EZH2 inhibitor is a small moleculedrug (e.g., tazemetostat or a tazemetostat salt) that is administereddaily (e.g., once a day, twice a day, three times a day, and so on)while the immune checkpoint inhibitor is a monoclonal antibody (e.g.,Nivolumab, Pembrolizumab, Atezolizumab, Durvalumab, or Avelumab) that isadministered at longer time intervals (e.g., once every two days, onceevery three days, once a week, once every two weeks, once every threeweeks, once a month, and so on).

In certain embodiments of the methods of the disclosure, the EZH2inhibitor and the immune checkpoint inhibitor are administeredsequentially. For example, the EZH2 inhibitor may be administered beforethe immune checkpoint inhibitor. Alternatively, the immune checkpointinhibitor may be administered before the EZH2 inhibitor. In someembodiments, the EZH2 inhibitor and the immune checkpoint inhibitor areadministered in temporal proximity, e.g., one is administrated withinone day, 12 hours, 6 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 15minutes, or 10 minutes before or after administration of the other. Incertain embodiments of the methods of the disclosure, the EZH2 inhibitorand the immune checkpoint inhibitor are administered simultaneously. Insome embodiments, the administration schedules of the EZH2 inhibitor andthe immune checkpoint inhibitor overlap. In some embodiments, thetreatment period of the EZH2 inhibitor and the immune checkpointinhibitor are the same, e.g., about four weeks, about five weeks, aboutsix weeks, about seven weeks, about eight weeks, about ten weeks, abouttwelve weeks, about fourteen weeks, about sixteen weeks, about eighteenweeks, or about 20 weeks. In some embodiments, the treatment period ofthe EZH2 inhibitor is longer than the treatment period of the immunecheckpoint inhibitor, or vice versa.

A therapeutically effective amount of a pharmaceutical agent providedherein, e.g., of an EZH2 inhibitor or an immune checkpoint inhibitor is,generally, an amount of the agent that is effective in treating,ameliorating, or preventing an identified disease or condition, or toexhibit a clinically desirable effect, e.g., a detectable therapeutic orinhibitory effect. In some embodiments, the effective amount is providedas weight of the pharmaceutical agent, e.g., of an EZH2 inhibitor or animmune checkpoint inhibitor provided herein, per body weight unit, e.g.,per kg body weight of the subject being administered the pharmaceuticalagent. In some embodiments, the effective amount is provided as a doseper day. Those of skill in the art will understand that an EZH2inhibitor and/or an immune checkpoint inhibitor provided herein may beadministered at a frequency other than once per day, e.g., twice perday, three times a day, once per week, once every two weeks, once everythree weeks, once per month, etc., and that the effective daily dose canbe determined by calculating the dose the patient receives per day,either cumulatively, where more than one dose is administered per day,or by dividing the total dose by the number of days in the dosageinterval (e.g., by 2 where a dose is administered every two days, by 3where a dose is administered every three days, etc.).

For example, in some embodiments, a therapeutically effective amount ofan EZH2 inhibitor and/or of an immune checkpoint inhibitor providedherein is between 1 μg (EZH2 inhibitor)/kg(body weight of the subject)and 1000 mg/kg, inclusive of the endpoints. In some embodiments, atherapeutically effective amount of the EZH2 inhibitor is between 1μg/kg and 1 mg/kg, 1 μg/kg and 10 mg/kg, 1 μg/kg and 25 mg/kg, 1 μg/kgand 50 mg/kg, 1 μg/kg and 100 mg/kg, 1 μg/kg and 250 mg/kg, 100 μg/kgand 50 mg/kg, 100 μg/kg and 500 mg/kg, 100 μg/kg and 1 mg/kg, 100 μg/kgand 10 mg/kg, 100 μg/kg and 25 mg/kg, 100 μg/kg and 50 mg/kg, 100 g/kgand 100 mg/kg, 100 μg/kg and 250 mg/kg, 1000 μg/kg and 5 mg/kg, 1000μg/kg and 10 mg/kg, 1000 μg/kg and 15 mg/kg, 1000 μg/kg and 20 mg/kg,1000 μg/kg and 25 mg/kg, 1000 g/kg and 50 mg/kg, 1000 μg/kg and 100mg/kg, 1000 μg/kg and 250 mg/kg, 1000 μg/kg and 500 mg/kg, 1000 μg/kgand 1000 mg/kg, 2500 μg/kg and 5 mg/kg, 2500 μg/kg and 10 mg/kg, 2500μg/kg and 15 mg/kg, 2500 μg/kg and 20 mg/kg, 2500 μg/kg and 25 mg/kg,2500 μg/kg and 50 mg/kg, 2500 μg/kg and 100 mg/kg, 2500 μg/kg and 250mg/kg, 2500 μg/kg and 500 mg/kg, 2500 μg/kg and 1000 mg/kg, 3000 μg/kgand 5 mg/kg, 5000 μg/kg and 10 mg/kg, 5000 g/kg and 15 mg/kg, 5000 μg/kgand 20 mg/kg, 10000 μg/kg and 25 mg/kg, 10000 μg/kg and 50 mg/kg, 10000μg/kg and 100 mg/kg, 10000 μg/kg and 250 mg/kg, 100000 μg/kg and 500mg/kg, 5 μg/kg and 500 mg/kg, 10 μg/kg and 500 mg/kg, 50 μg/kg and 500mg/kg, 100 μg/kg and 500 mg/kg, 250 μg/kg and 500 mg/kg, 500 μg/kg and500 mg/kg, 1000 μg/kg and 500 mg/kg, 5 μg/kg and 100 mg/kg, 10 μg/kg and100 mg/kg, 50 μg/kg and 100 mg/kg, 100 μg/kg and 100 mg/kg, 250 μg/kgand 100 mg/kg, 500 μg/kg and 100 mg/kg, 1000 μg/kg and 100 mg/kg, 5μg/kg and 10 mg/kg, 10 μg/kg and 10 mg/kg, 50 μg/kg and 10 mg/kg, 100μg/kg and 10 mg/kg, 250 μg/kg and 10 mg/kg, 500 μg/kg and 10 mg/kg, 750μg/kg and 10 mg/kg, 1000 μg/kg and 10 mg/kg, 5 μg/kg and 1 mg/kg, 10μg/kg and 1 mg/kg, 50 μg/kg and 1 mg/kg, 100 μg/kg and 1 mg/kg, 250μg/kg and 1 mg/kg, 500 μg/kg and 1 mg/kg, 750 μg/kg and 1 mg/kg, and 750μg/kg and 1.5 mg/kg inclusive of the endpoints. In some embodiments, atherapeutically effective amount of an EZH2 inhibitor and/or of animmune checkpoint inhibitor provided herein is between 1 μg(EZH2inhibitor)/kg(body weight of the subject)/day and 1000 mg/kg/day,inclusive of the endpoints. In some embodiments, a therapeuticallyeffective amount of the EZH2 inhibitor is between 1 μg/kg/day and 1mg/kg/day, 1 μg/kg/day and 10 mg/kg/day, 1 μg/kg/day and 25 mg/kg/day, 1μg/kg/day and 50 mg/kg/day, 1 μg/kg/day and 100 mg/kg/day, 1 μg/kg/dayand 250 mg/kg/day, 100 μg/kg/day and 50 mg/kg/day, 100 μg/kg/day and 500mg/kg/day, 100 μg/kg/day and 1 mg/kg/day, 100 μg/kg/day and 10mg/kg/day, 100 μg/kg/day and 25 mg/kg/day, 100 μg/kg/day and 50mg/kg/day, 100 μg/kg/day and 100 mg/kg/day, 100 μg/kg/day and 250mg/kg/day, 1000 μg/kg/day and 5 mg/kg/day, 1000 ag/kg/day and 10mg/kg/day, 1000 μg/kg/day and 15 mg/kg/day, 1000 μg/kg/day and 20mg/kg/day, 1000 μg/kg/day and 25 mg/kg/day, 1000 μg/kg/day and 50mg/kg/day, 1000 μg/kg/day and 100 mg/kg/day, 1000 μg/kg/day and 250mg/kg/day, 1000 μg/kg/day and 500 mg/kg/day, 1000 μg/kg/day and 1000mg/kg/day, 2500 μg/kg/day and 5 mg/kg/day, 2500 μg/kg/day and 10mg/kg/day, 2500 μg/kg/day and 15 mg/kg/day, 2500 μg/kg/day and 20mg/kg/day, 2500 μg/kg/day and 25 mg/kg/day, 2500 μg/kg/day and 50mg/kg/day, 2500 μg/kg/day and 100 mg/kg/day, 2500 μg/kg/day and 250mg/kg/day, 2500 μg/kg/day and 500 mg/kg/day, 2500 μg/kg/day and 1000mg/kg/day, 3000 μg/kg/day and 5 mg/kg/day, 5000 μg/kg/day and 10mg/kg/day, 5000 μg/kg/day and 15 mg/kg/day, 5000 μg/kg/day and 20mg/kg/day, 10000 μg/kg/day and 25 mg/kg/day, 10000 μg/kg/day and 50mg/kg/day, 10000 μg/kg/day and 100 mg/kg/day, 10000 μg/kg/day and 250mg/kg/day, 100000 μg/kg/day and 500 mg/kg/day, 5 μg/kg/day and 500mg/kg/day, 10 μg/kg/day and 500 mg/kg/day, 50 μg/kg/day and 500mg/kg/day, 100 μg/kg/day and 500 mg/kg/day, 250 μg/kg/day and 500mg/kg/day, 500 μg/kg/day and 500 mg/kg/day, 1000 μg/kg/day and 500mg/kg/day, 5 μg/kg/day and 100 mg/kg/day, 10 μg/kg/day and 100mg/kg/day, 50 μg/kg/day and 100 mg/kg/day, 100 μg/kg/day and 100mg/kg/day, 250 μg/kg/day and 100 mg/kg/day, 500 μg/kg/day and 100mg/kg/day, 1000 μg/kg/day and 100 mg/kg/day, 5 μg/kg/day and 10mg/kg/day, 10 μg/kg/day and 10 mg/kg/day, 50 μg/kg/day and 10 mg/kg/day,100 μg/kg/day and 10 mg/kg/day, 250 μg/kg/day and 10 mg/kg/day, 500μg/kg/day and 10 mg/kg/day, 750 ag/kg/day and 10 mg/kg/day, and 1000μg/kg/day, 10 mg/kg/day, 5 μg/kg/day and 1 mg/kg/day, 10 μg/kg/day and 1mg/kg/day, 50 μg/kg/day and 1 mg/kg/day, 100 μg/kg/day and 1 mg/kg/day,250 μg/kg/day and 1 mg/kg/day, 500 μg/kg/day and 1 mg/kg/day, 750μg/kg/day and 1 mg/kg/day, and 750 μg/kg/day and 1.5 mg/kg/day,inclusive of the endpoints. In some embodiments, an effective amount ofan EZH2 inhibitor and/or of an immune checkpoint inhibitor providedherein is about 1 μg/kg, about 2 μg/kg, about 2.5 μg/kg, about 5 μg/kg,about 10 μg/kg, about 20 μg/kg, about 25 μg/kg, about 50 μg/kg, about100 μg/kg, about 200 μg/kg, about 250 μg/kg, about 500 μg/kg, about 1mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 5 mg/kg, about 10 mg/kg,about 20 mg/kg, about 25 mg/kg, about 50 mg/kg, about 100 mg/kg, about200 mg/kg, about 250 mg/kg, about 500 mg/kg, or about 1000 mg/kg. Insome embodiments, an effective amount of an EZH2 inhibitor and/or of animmune checkpoint inhibitor provided herein is about 1 μg/kg/day, about2 μg/kg/day, about 2.5 μg/kg/day, about 5 μg/kg/day, about 10 μg/kg/day,about 20 μg/kg/day, about 25 μg/kg/day, about 50 μg/kg/day, about 100μg/kg/day, about 200 μg/kg/day, about 250 μg/kg/day, about 500μg/kg/day, about 1 mg/kg/day, about 2 mg/kg/day, about 2.5 mg/kg/day,about 5 mg/kg/day, about 10 mg/kg/day, about 20 mg/kg/day, about 25mg/kg/day, about 50 mg/kg/day, about 100 mg/kg/day, about 200 mg/kg/day,about 250 mg/kg/day, about 500 mg/kg/day, or about 1000 mg/kg/day. Thedisclosure embraces methods and treatment strategies using anycombination of EZH2 inhibitors and immune checkpoint inhibitors providedherein at any dosage, combination of dosages, administration routes, anddosage intervals, provided herein. For example, in some embodiments, theEZH2 inhibitor (e.g., tazemetostat) is administered at a dosage of about10 mg/kg per day by administration twice a day, and the immunecheckpoint inhibitor (e.g., a monoclonal antibody, such as, for example,Nivolumab, Pembrolizumab, Atezolizumab, Durvalumab, or Avelumab) isadministered at a dosage of 500 μg/kg/day and 1 mg/kg/day byadministration once every three weeks.

In some embodiments, the EZH2 inhibitor and/or the immune checkpointinhibitor is administered in a solid or liquid formulation, e.g., in apill, a tablet, a solution, or a suspension. In some embodiments, atherapeutically effective amount of the EZH2 inhibitor and/or of theimmune checkpoint inhibitor, e.g., an effective amount as providedherein, is administered to a subject in a formulation volume between 1μL and 500 mL, inclusive of the endpoints. In some embodiments, atherapeutically effective amount of the EZH2 inhibitor and/or of theimmune checkpoint inhibitor, e.g., an effective amount as providedherein is administered in a formulation volume between 1 mL and 500 mL,1 mL and 200 mL, 1 mL and 20 mL, 1 mL and 10 mL, 1 mL and 5 mL, 0.5 mLand 5 mL, 0.5 mL and 2 mL, 0.1 mL and 1 mL, or 0.1 mL and 0.5 mL,inclusive of the endpoints. In some embodiments where the EZH2 inhibitorand the immune checkpoint inhibitor are administrated in separateformulations to the subject, any combination of such volumes may beused. For example, the EZH2 inhibitor may be administrated orally in a100 mL suspension, and the checkpoint inhibitor may be administrated byinjection of a 1 mL liquid formulation.

In certain embodiments, the EZH2 inhibitor and/or the immune checkpointinhibitor is administered systemically. In some embodiments, the EZH2inhibitor and/or the immune checkpoint inhibitor is administered via anoral or a parenteral route. In some embodiments, the EZH2 inhibitor andthe immune checkpoint inhibitor are administered via different routes,e.g., one is administered orally and the other parenterally. In certainembodiments, the EZH2 inhibitor is administered orally, e.g., formulatedas a capsule, tablet, suspension, or solution for oral administration.In certain embodiments, the immune checkpoint inhibitor is administeredvia a parenteral route. In some embodiments, the EZH2 inhibitor may beformulated as a solid or liquid, e.g., as a pill, tablet, solution, orsuspension, for oral administration and the immune checkpoint inhibitoris formulated as a liquid, e.g., a solution or suspension, forparenteral administration, e.g., for intravenous injection.

In certain embodiments of the methods of this disclosure, the EZH2inhibitor is administered twice a day at a dosage of 800 mg and theimmune checkpoint inhibitor is administered once every three weeks at adosage of 1200 mg. In certain aspects, the EZH2 inhibitor is a smallmolecule drug and the immune checkpoint inhibitor is a monoclonalantibody. In certain aspects, the EZH2 inhibitor is administered orally.In certain aspects, the EZH2 inhibitor is administered orally and theimmune checkpoint inhibitor is administered parenterally. In certainaspects, the immune checkpoint inhibitor comprises a PD-L1 inhibitor. Incertain aspects, the immune checkpoint inhibitor comprises Atezolizumab.In certain aspects, the immune checkpoint inhibitor comprises Nivolumab.In certain aspects, the immune checkpoint inhibitor comprisesPembrolizumab.

It will be understood that the effective amounts, formulation volumes,and administration routes provided herein are non-limiting examples ofsome embodiments within the scope of this disclosure. Additionalsuitable amounts and administration routes will be apparent to theperson of ordinary skill in the art based on this disclosure and thegeneral knowledge in the art. The present disclosure is not limited inthis respect. In some embodiments, the subject being administered theEZH2 inhibitor and the immune checkpoint inhibitor has or is diagnosedwith a proliferative disease. In some embodiments, the proliferativedisease is a malignant proliferative disease, e.g., a cancer. In someembodiments, a cancer that can be treated by the methods of thedisclosure or with the compositions, strategies, treatment modalities,methods, combinations, and compositions of the disclosure comprises oris derived from a stem cell or a progenitor cell.

In some embodiments, tissue biopsies after the subject is administeredthe EZH2 inhibitor show an increase of expression of an immunecheckpoint protein (e.g., PD-L1) as compared to a reference level. Insome embodiments, the reference level is the level of expression of theimmune checkpoint protein observed in tissue biopsies collected prior toadministration of the EZH2 inhibitor. In some embodiments, theexpression of the immune checkpoint protein is increased by 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%,600%, 700%, 800%, 900%, or 1000%, as compared to the reference level. Insome embodiments, the subject administered the EZH2 inhibitor is notreceiving an immune checkpoint inhibitor prior to administration of theEZH2 inhibitor. In some embodiments, the subject administered the EZH2inhibitor is receiving an immune checkpoint inhibitor prior toadministration of the EZH2 inhibitor.

In some embodiments, cancers that can be treated by the methods of thedisclosure or with the compositions, strategies, treatment modalities,methods, combinations, and compositions of the disclosure comprise orare derived from an immune cell. In some embodiments, the cancer is aform of soft tissue sarcoma, e.g., epithelioid sarcoma or clear cellsarcoma of soft tissue.

In some embodiments, cancers that can be treated by the methods of thedisclosure or with the compositions, strategies, treatment modalities,methods, combinations, and compositions of the disclosure comprise orare derived adrenocortical carcinoma, AIDS-related cancers, AIDS-relatedlymphoma, anal cancer, anorectal cancer, cancer of the anal canal,appendix cancer, childhood cerebellar astrocytoma, childhood cerebralastrocytoma, basal cell carcinoma, skin cancer (non-melanoma), biliarycancer, extrahepatic bile duct cancer, intrahepatic bile duct cancer,bladder cancer, urinary bladder cancer, bone and joint cancer,osteosarcoma and malignant fibrous histiocytoma, brain cancer, braintumor, brain stem glioma, cerebellar astrocytoma, cerebralastrocytoma/malignant glioma, ependymoma, medulloblastoma,supratentorial primitive neuroectodermal tumors, visual pathway andhypothalamic glioma, breast cancer, bronchial adenomas/carcinoids,carcinoid tumor, gastrointestinal, nervous system cancer, nervous systemlymphoma, central nervous system cancer, central nervous systemlymphoma, cervical cancer, childhood cancers, chronic lymphocyticleukemia, chronic myelogenous leukemia, chronic myeloproliferativedisorders, colon cancer, colorectal cancer, cutaneous T-cell lymphoma,lymphoid neoplasm, mycosis fungoides, Sezary Syndrome, endometrialcancer, esophageal cancer, extracranial germ cell tumor, extragonadalgerm cell tumor, extrahepatic bile duct cancer, eye cancer, intraocularmelanoma, retinoblastoma, gallbladder cancer, gastric (stomach) cancer,gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST),germ cell tumor, ovarian germ cell tumor, gestational trophoblastictumor glioma, head and neck cancer, hepatocellular (liver) cancer,Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, ocularcancer, islet cell tumors (endocrine pancreas), Kaposi Sarcoma, kidneycancer, renal cancer, kidney cancer, laryngeal cancer, acutelymphoblastic leukemia, acute myeloid leukemia, chronic lymphocyticleukemia, chronic myelogenous leukemia, hairy cell leukemia, lip andoral cavity cancer, liver cancer, lung cancer, non-small cell lungcancer, small cell lung cancer, AIDS-related lymphoma, non-Hodgkinlymphoma, primary central nervous system lymphoma, Waldenstroemmacroglobulinemia, melanoma, intraocular (eye) melanoma, merkel cellcarcinoma, mesothelioma malignant, mesothelioma, metastatic squamousneck cancer, mouth cancer, cancer of the tongue, multiple endocrineneoplasia syndrome, mycosis fungoides, myelodysplastic syndromes,myelodysplastic/myeloproliferative diseases, chronic myelogenousleukemia, acute myeloid leukemia, multiple myeloma, chronicmyeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, oralcancer, oral cavity cancer, oropharyngeal cancer, ovarian cancer,ovarian epithelial cancer, ovarian low malignant potential tumor,ovarian clear cell adenocarcinoma, ovarian endometrioid adenocarcinoma,ovarian serous adenocarcinoma, pancreatic ductal adenocarcinoma,pancreatic endocrine tumor, pancreatic cancer, islet cell pancreaticcancer, paranasal sinus and nasal cavity cancer, parathyroid cancer,penile cancer, pharyngeal cancer, pheochromocytoma, pineoblastoma andsupratentorial primitive neuroectodermal tumors, pituitary tumor, plasmacell neoplasm/multiple myeloma, pleuropulmonary blastoma, prostatecancer, rectal cancer, renal pelvis and ureter, transitional cellcancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, ewingfamily of sarcoma tumors, Kaposi Sarcoma, soft tissue sarcoma, synovialsarcoma, uterine cancer, uterine sarcoma, skin cancer (non-melanoma),skin cancer (melanoma), merkel cell skin carcinoma, small intestinecancer, soft tissue sarcoma, squamous cell carcinoma, stomach (gastric)cancer, supratentorial primitive neuroectodermal tumors, testicularcancer, throat cancer, thymoma, thymoma and thymic carcinoma, thyroidcancer, transitional cell cancer of the renal pelvis and ureter andother urinary organs, gestational trophoblastic tumor, urethral cancer,endometrial uterine cancer, uterine sarcoma, uterine corpus cancer,vaginal cancer, vulvar cancer, Wilm's Tumor, malignant rhabdoid tumor,astrocytoma, atypical teratoid rhabdoid tumor, choroid plexus carcinoma,choroid plexus papilloma, ependymoma, glioblastoma, meningioma,neuroglial tumor, oligoastrocytoma, oligodendroglioma, carcinosarcoma,chordoma, extrarenal rhabdoid tumor, schwannoma, skin squamous cellcarcinoma, chondrosarcoma, clear cell sarcoma of soft tissue, ewingsarcoma, epithelioid sarcoma, renal medullary carcinoma, diffuse largeB-cell lymphoma, follicular lymphoma and not otherwise specified (NOS)sarcoma.

In some embodiments, the cancer is an INI1-negative tumor. INI1 (alsocalled SNF5, SMARCB1 or BAF47), is a critical component of the SWI/SNFregulatory complex, a chromatin remodeler that acts in opposition toEZH2. INI1-negative tumors have altered SWI/SNF function, resulting inaberrant and oncogenic EZH2 activity. This activity can be targeted bysmall molecule inhibitors of EZH2 such as tazemetostat. INI1-negativetumors are generally aggressive and are poorly served by currenttreatments. For example, current treatment of MRT, a well-studiedINI1-negative tumor, consists of surgery, chemotherapy and radiationtherapy, which are associated with limited efficacy and significanttreatment-related morbidity. Non-limiting examples of INI1-negativetumors are rhabdoid tumors of the kidney (RTK), atypicalteratoid/rhabdoid tumors (ATRT), epithelioid malignant peripheral nervesheath tumors, myoepithelial carcinoma, and renal medullary carcinoma.

In some embodiments, a cancer that can be treated with the strategies,treatment modalities, methods, combinations, and compositions of thedisclosure comprise a solid tumor. In some embodiments, a cancer thatcan be treated with the strategies, treatment modalities, methods,combinations, and compositions of the disclosure comprises or is derivedfrom a cell of epithelial origin. In some embodiments, cancers that canbe treated with the strategies, treatment modalities, methods,combinations, and compositions of the disclosure are primary tumors. Insome embodiments, cancers that can be treated with the strategies,treatment modalities, methods, combinations, and compositions of thedisclosure are secondary tumors. In some embodiments, the cancer ismetastatic.

In certain embodiments of the methods of the disclosure, the subjectbeing administered the EZH2 inhibitor and the immune checkpointinhibitor has been diagnosed with cancer. In some embodiments, thesubject is an adult. In some embodiments, the subject is a pediatricsubject. In some embodiments, the subject is a human.

In certain embodiments, the subject is an adult, and the therapeuticallyeffective amount of tazemetostat is about 100 mg to about 1600 mg. Incertain embodiments, the subject is an adult, and the therapeuticallyeffective amount of tazemetostat is about 100 mg, 200 mg, 400 mg, 800mg, or about 1600 mg. In certain embodiments, the subject is an adult,and the therapeutically effective amount of tazemetostat is about 800mg, e.g., 800 mg/day administered at a dose of 400 mg orally twice aday.

In certain embodiments, the subject is pediatric, and the tazemetostatmay be administered at a dose of between 230 mg/m² and 600 mg/m² twiceper day (BID), inclusive of the endpoints. In certain embodiments, thesubject is pediatric, and the tazemetostat is administered at a dose ofbetween 230 mg/m² and 305 mg/m² twice per day (BID), inclusive of theendpoints. In certain embodiments, the subject is pediatric, and thetazemetostat is administered at a dose of 240 mg/m² twice per day (BID).In certain embodiments, the subject is pediatric, and the tazemetostatis administered at a dose of 300 mg/m² twice per day (BID). In certainembodiments, the subject is pediatric, and the tazemetostat isadministered at a dose of about 60% of the area under the curve (AUC) atsteady state (AUC_(SS)) following administration of 1600 mg twice a dayto an adult subject. In certain embodiments, the subject is pediatric,and the tazemetostat is administered at a dose of about 600 mg/m² perday. In certain embodiments, the subject is pediatric, and thetazemetostat is administered at a dose of at least 600 mg/m² per day. Incertain embodiments, the subject is pediatric, and the tazemetostat isadministered at a dose of about 80% of the area under the curve (AUC) atsteady state (AUC_(SS)) following administration of 800 mg twice a dayto an adult subject. In certain embodiments, the subject is pediatric,and tazemetostat is administered at a dose of about 390 mg/m² twice perday (BID). In certain embodiments, the subject is pediatric, and thetazemetostat is administered at a dose of at least 390 mg/m² twice perday (BID). In certain embodiments, the subject is pediatric, and thetazemetostat is administered at a dose of between 300 mg/m² and 600mg/m² twice per day (BID).

In some embodiments, e.g., in some embodiments where the subject ispediatric, the EZH2 inhibitor may be formulated as an oral suspension.

In certain embodiments, this disclosure provides a method ofadministering to a subject having soft tissue sarcoma (or any other formof cancer) a combination of tazemetostat at an oral dose of 800 mg twiceper day and atezolizumab (TECENTRIQ™) at a dose of 1200 mg as anintravenous infusion over 60 minutes every 3 weeks (see,accessdata.fda.gov/drugsatfda_docs/label/2016/761034s0001b1.pdf, thecontents of which are incorporated herein for additional informationabout atezolizumab).

In certain embodiments, this disclosure provides a method ofadministering to a subject having epithelioid sarcoma (or any other formof cancer) a combination of tazemetostat at an oral dose of 800 mg twiceper day and atezolizumab (TECENTRIQ™) at a dose of 1200 mg as anintravenous infusion over 60 minutes every 3 weeks.

In certain embodiments, this disclosure provides a method ofadministering to a subject having locally advanced or metastaticurothelial carcinoma (or any other form of cancer) a combination oftazemetostat at an oral dose of 800 mg twice per day and atezolizumab(TECENTRIQ™) at a dose of 1200 mg as an intravenous infusion over 60minutes every 3 weeks.

In certain embodiments, this disclosure provides a method ofadministering to a subject having Diffuse Large B-Cell Lymphoma (DLBCL)(or any other form of cancer) a combination of tazemetostat at an oraldose of 800 mg twice per day and atezolizumab (TECENTRIQ™) at a dose of1200 mg as an intravenous infusion over 60 minutes every 3 weeks.

In certain embodiments, this disclosure provides a method ofadministering to a subject having Non-Hodgkin's Lymphoma (or any otherform of cancer) a combination of tazemetostat at an oral dose of 800 mgtwice per day and atezolizumab (TECENTRIQ™) at a dose of 1200 mg as anintravenous infusion over 60 minutes every 3 weeks.

In certain embodiments, this disclosure provides a method ofadministering to a subject having soft tissue sarcoma (or any other formof cancer) a combination of tazemetostat at an oral dose of 800 mg twiceper day and nivolumab (OPTIVO™) at a dose of 3 mg/kg as an intravenousinfusion over 60 minutes every 2 weeks (see,accessdata.fda.gov/drugsatfda_docs/label/2014/1255541b1.pdf, thecontents of which are incorporated herein for additional informationabout nivolumab).

In certain embodiments, this disclosure provides a method ofadministering to a subject having epithelioid sarcoma (or any other formof cancer) a combination of tazemetostat at an oral dose of 800 mg twiceper day and nivolumab (OPTIVO™) at a dose of 3 mg/kg as an intravenousinfusion over 60 minutes every 2 weeks.

In certain embodiments, this disclosure provides a method ofadministering to a subject having melanoma (or any other form of cancer)a combination of tazemetostat at an oral dose of 800 mg twice per dayand nivolumab (OPTIVO™) at a dose of 3 mg/kg as an intravenous infusionover 60 minutes every 2 weeks.

In certain embodiments, this disclosure provides a method ofadministering to a subject having melanoma (or any other form of cancer)and having a BRAF V600 mutation a combination of tazemetostat at an oraldose of 800 mg twice per day and nivolumab (OPTIVO™) at a dose of 3mg/kg as an intravenous infusion over 60 minutes every 2 weeks, andoptionally, a BRAF inhibitor.

In certain embodiments, this disclosure provides a method ofadministering to a subject having soft tissue sarcoma (or any other formof cancer) a combination of tazemetostat at an oral dose of 800 mg twiceper day and pembrolizumab (KEYTRUDA™) at a dose of 2 mg/kg as anintravenous infusion over 30 minutes every 3 weeks (see,accessdata.fda.gov/drugsatfda_docs/label/2014/1255141b1.pdf, thecontents of which are incorporated herein for additional informationabout pembrolizumab).

In certain embodiments, this disclosure provides a method ofadministering to a subject having epithelioid sarcoma (or any other formof cancer) a combination of tazemetostat at an oral dose of 800 mg twiceper day and pembrolizumab (KEYTRUDA™) at a dose of 2 mg/kg as anintravenous infusion over 30 minutes every 3 weeks.

In certain embodiments, this disclosure provides a method ofadministering to a subject having melanoma and disease progressionfollowing ipilimumab (or any other form of cancer) a combination oftazemetostat at an oral dose of 800 mg twice per day and pembrolizumab(KEYTRUDA™) at a dose of 2 mg/kg as an intravenous infusion over 30minutes every 3 weeks.

In certain embodiments, this disclosure provides a method ofadministering to a subject having unresectable or metastatic melanomaand disease progression following ipilimumab (or any other form ofcancer) and having a BRAF V600 mutation a combination of tazemetostat atan oral dose of 800 mg/kg twice per day and pembrolizumab (KEYTRUDA™) ata dose of 2 mg/kg as an intravenous infusion over 30 minutes every 3weeks, and optionally, a BRAF inhibitor.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. In the specification, thesingular forms also include the plural unless the context clearlydictates otherwise. All publications, patent applications, patents andother references mentioned herein are incorporated by reference. Thereferences cited herein are not admitted to be prior art to the claimedinvention. In the case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods andexamples are illustrative only and are not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A is a PD-L1 stain of a pre-dose tissue biopsy of tissue takenfrom the left upper arm of a subject with epithelioid sarcoma.

FIG. 1B is a PD-L1 stain of a post-dose tissue biopsy of tissue takenfrom the left upper arm of a subject with epithelioid sarcoma. Thetissue was taken after administration of tazemetostat at 800 mg (b.i.d.)for 25 weeks.

FIG. 2A is a CD4 stain of a pre-dose tissue biopsy of tissue taken fromthe left upper arm of a subject with epithelioid sarcoma.

FIG. 2B is a CD4 stain of a post-dose tissue biopsy of tissue taken fromthe left upper arm of a subject with epithelioid sarcoma. The tissue wastaken after administration of tazemetostat at 800 mg (b.i.d.) for 25weeks.

FIG. 3A is a CD8 stain of a pre-dose tissue biopsy of tissue taken fromthe left upper arm of a subject with epithelioid sarcoma.

FIG. 3B is a CD8 stain of a post-dose tissue biopsy of tissue taken fromthe left upper arm of a subject with epithelioid sarcoma. The tissue wastaken after administration of tazemetostat at 800 mg (b.i.d.) for 25weeks.

FIG. 4A is a PD-L1 stain of a pre-dose tissue biopsy of tissue takenfrom a subject with renal medullary carcinoma.

FIG. 4B is a PD-L1 stain of a post-dose tissue biopsy of tissue takenfrom a subject with renal medullary carcinoma. The tissue was takenafter administration of tazemetostat for 8 weeks.

FIG. 5A is a CD8+ stain of a pre-dose tissue biopsy of tissue taken froma subject with renal medullary carcinoma.

FIG. 5B is a CD8+ stain of a post-dose tissue biopsy of tissue takenfrom a subject with renal medullary carcinoma. The tissue was takenafter administration of tazemetostat for 8 weeks.

DETAILED DESCRIPTION

Some aspects of the disclosure provide methods comprising detectingexpression of an immune checkpoint protein in a subject having cancer.In some embodiments, the subject has been administered an effectiveamount of an anti-cancer drug, e.g., of an enhancer of zeste homolog 2(EZH2) inhibitor. Some aspects of the disclosure relate to methodscomprising administering to a subject having a cancer expressing animmune checkpoint protein, e.g., a cancer showing expression and/orupregulation of the immune checkpoint protein after treatment with ananti-cancer drug, an effective amount of an EZH2 inhibitor and aneffective amount of an immune checkpoint inhibitor.

EZH2

EZH2 is a histone methyltransferase that is the catalytic subunit of thePRC2 complex which catalyzes the mono- through tri-methylation of lysine27 on histone H3 (H3-K27). Histone H3-K27 trimethylation is a mechanismfor suppressing transcription of specific genes that are proximal to thesite of histone modification. This trimethylation is known to be acancer marker with altered expression in cancer, such as prostate cancer(see, e.g., U.S. Patent Application Publication No. 2003/0175736;incorporated herein by reference in its entirety). Other studiesprovided evidence for a functional link between dysregulated EZH2expression, transcriptional repression, and neoplastic transformation.Varambally et al. (2002) Nature 419(6907):624-9 Kleer et al. (2003) ProcNatl Acad Sci USA 100(20):11606-11; incorporated herein by reference inits entirety.

Human EZH2 nucleic acids and polypeptides have previously beendescribed. See, e.g., Chen et al. (1996) Genomics 38:30-7 [746 aminoacids]; Swiss-Prot Accession No. Q15910 [746 amino acids]; GenBankAccession Nos. NM_004456 and NP_004447 (isoform a [751 amino acids]);and GenBank Accession Nos. NM_152998 and NP_694543 (isoform b [707 aminoacids]), each of which is incorporated herein by reference in itsentirety.

Some aspects of the disclosure provide methods for treating oralleviating a symptom of cancer or precancerous condition in a subjectby administering to a subject expressing either a wild type or a mutantEZH2 a therapeutically effective amount of an EZH2 inhibitor and animmune checkpoint modulator as described herein. In certain embodiments,the EZH2 inhibitor is tazemetostat or a pharmaceutically acceptable saltthereof.

Some aspects of this disclosure provide methods for inhibiting in asubject conversion of H3-K27 to trimethylated H3-K27, while alsoinhibiting an immune checkpoint in the subject. The inhibition of theconversion from H3-K27 to trimethylated H3-K27 involves, in someembodiments, inhibiting in a subject conversion of unmethylated H3-K27to monomethylated H3-K27, conversion of monomethylated H3-K27 todimethylated H3-K27, conversion of dimethylated H3-K27 to trimethylatedH3-K27, or any combination thereof, including, for example, conversionof monomethylated H3-K27 to dimethylated H3-K27 and conversion ofdimethylated H3-K27 to trimethylated H3-K27. As used herein,unmethylated H3-K27 refers to histone H3 with no methyl group covalentlylinked to the amino group of lysine 27. As used herein, monomethylatedH3-K27 refers to histone H3 with a single methyl group covalently linkedto the amino group of lysine 27. Monomethylated H3-K27 is also referredto herein as H3-K27me1. As used herein, dimethylated H3-K27 refers tohistone H3 with two methyl groups covalently linked to the amino groupof lysine 27. Dimethylated H3-K27 is also referred to herein asH3-K27me2. As used herein, trimethylated H3-K27 refers to histone H3with three methyl groups covalently linked to the amino group of lysine27. Trimethylated H3-K27 is also referred to herein as H3-K27me3.

EZH2 Inhibitors

Various small molecule EZH2 inhibitors have previously been described.Some non-limiting examples of EZH2 inhibitors that are suitable for usein the strategies, treatment modalities, methods, combinations, andcompositions provided herein are those described in U.S. Pat. Nos.8,410,088, 8,765,732, 9,090,562, 8,598,167, 8,962,620, US-2015/0065483,U.S. Pat. Nos. 9,206,157, 9,006,242, 9,089,575, US 2015-0352119, WO2014/062733, US-2015/0065503, WO2015/057859, U.S. Pat. No. 8,536,179, WO2011/140324, PCT/US2014/015706, published as WO/2014/124418, inPCT/US2013/025639, published as WO/2013/120104, and in U.S. Ser. No.14/839,273, published as US 2015/0368229, the entire contents of each ofwhich are incorporated herein by reference.

In some embodiments, an EZH2 inhibitor suitable for use in thestrategies, treatment modalities, methods, combinations, andcompositions described herein has the following Formula (I):

or a pharmaceutically acceptable salt thereof; wherein

R⁷⁰¹ is H, F, OR⁷⁰⁷, NHR⁷⁰⁷, —(C≡C)—(CH₂)_(n7)—R⁷⁰⁸, phenyl, 5- or6-membered heteroaryl, C₃₋₈ cycloalkyl, or 4-7 membered heterocycloalkylcontaining 1-3 heteroatoms, wherein the phenyl, 5- or 6-memberedheteroaryl, C₃₋₈ cycloalkyl or 4-7 membered heterocycloalkyl eachindependently is optionally substituted with one or more groups selectedfrom halo, C₁₋₃ alkyl, OH, O—C₁₋₆ alkyl, NH—C₁₋₆ alkyl, and, C₁₋₃ alkylsubstituted with C₃₋₈ cycloalkyl or 4-7 membered heterocycloalkylcontaining 1-3 heteroatoms, wherein each of the O—C₁₋₆ alkyl and NH—C₁₋₆alkyl is optionally substituted with hydroxyl, O—C₁₋₃ alkyl or NH—C₁₋₃alkyl, each of the O—C₁₋₃ alkyl and NH—C₁₋₃ alkyl being optionallyfurther substituted with O—C₁₋₃ alkyl or NH—C₁₋₃ alkyl;

each of R⁷⁰² and R⁷⁰³, independently is H, halo, C₁₋₄ alkyl, C₁₋₆alkoxyl or C₆-C₁₀ aryloxy, each optionally substituted with one or morehalo;

each of R⁷⁰⁴ and R⁷⁰⁵, independently is C₁₋₄ alkyl;

R⁷⁰⁶ is cyclohexyl substituted by N(C₁₋₄ alkyl)₂ wherein one or both ofthe C₁₋₄ alkyl is optionally substituted with C₁₋₆ alkoxy; or R⁷⁰⁶ istetrahydropyranyl;

R⁷⁰⁷ is C₁₋₄ alkyl optionally substituted with one or more groupsselected from hydroxyl, C₁₋₄ alkoxy, amino, mono- or di-C₁₋₄ alkylamino,C₃₋₈ cycloalkyl, and 4-7 membered heterocycloalkyl containing 1-3heteroatoms, wherein the C₃₋₈ cycloalkyl or 4-7 memberedheterocycloalkyl each independently is further optionally substitutedwith C₁₋₃ alkyl;

R⁷⁰⁸ is C₁₋₄ alkyl optionally substituted with one or more groupsselected from OH, halo, and C₁₋₄ alkoxy, 4-7 membered heterocycloalkylcontaining 1-3 heteroatoms, or O—C₁₋₆ alkyl, wherein the 4-7 memberedheterocycloalkyl can be optionally further substituted with OH or C₁₋₆alkyl; and

n₇ is 0, 1 or 2.

For example, R⁷⁰⁶ is cyclohexyl substituted by N(C₁₋₄ alkyl)₂ whereinone of the C₁₋₄ alkyl is unsubstituted and the other is substituted withmethoxy.

For example, R⁷⁰⁶ is

For example, the compound is of Formula II.

For example, R⁷⁰² is methyl or isopropyl and R⁷⁰³ is methyl or methoxyl.

For example, R⁷⁰⁴ is methyl.

For example, R⁷⁰¹ is OR⁷⁰⁷ and R⁷⁰⁷ is C₁₋₃ alkyl optionally substitutedwith OCH₃ or morpholine.

For example, R⁷⁰¹ is H or F.

For example, R⁷⁰¹ is tetrahydropyranyl, phenyl, pyridyl, pyrimidyl,pyrazinyl, imidazolyl, or pyrazolyl, each of which is optionallysubstituted with methyl, methoxy, ethyl substituted with morpholine, or—OCH₂CH₂OCH₃.

For example, R⁷⁰⁸ is morpholine, piperidine, piperazine, pyrrolidine,diazepane, or azetidine, each of which is optionally substituted with OHor C₁₋₆ alkyl.

For example, R⁷⁰⁸ is morpholine

For example, R⁷⁰⁸ is piperazine substituted with C₁₋₆ alkyl.

For example, R⁷⁰⁸ is methyl, t-butyl or C(CH₃)₂OH.

In some embodiments, an EZH2 inhibitor that can be used in thestrategies, treatment modalities, methods, combinations, andcompositions described herein may have the following Formula III:

or a pharmaceutically acceptable salt thereof.

In this formula:

R⁸⁰¹ is C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₈ cycloalkyl, 4-7membered heterocycloalkyl containing 1-3 heteroatoms, phenyl or 5- or6-membered heteroaryl, each of which is substituted with O—C₁₋₆alkyl-R_(x) or NH—C₁₋₆ alkyl-R_(x), wherein R_(x) is hydroxyl, O—C₁₋₃alkyl or NH—C₁₋₃ alkyl, and R_(x) is optionally further substituted withO—C₁₋₃ alkyl or NH—C₁₋₃ alkyl except when R_(x) is hydroxyl; or R⁸⁰¹ isphenyl substituted with -Q₂-T₂, wherein Q₂ is a bond or C₁-C₃ alkyllinker optionally substituted with halo, cyano, hydroxyl or C₁-C₆alkoxy, and T₂ is optionally substituted 4- to 12-memberedheterocycloalkyl; and R⁸⁰¹ is optionally further substituted;

each of R⁸⁰² and R⁸⁰³, independently is H, halo, C₁₋₄ alkyl, C₁₋₆alkoxyl or C₆-C₁₀ aryloxy, each optionally substituted with one or morehalo;

each of R⁸⁰⁴ and R⁸⁰⁵, independently is C₁₋₄ alkyl; and

R⁸⁰⁶ is -Q_(x)-T_(x), wherein Q_(x) is a bond or C₁₋₄ alkyl linker,T_(x) is H, optionally substituted C₁₋₄ alkyl, optionally substitutedC₃-C₈ cycloalkyl or optionally substituted 4- to 14-memberedheterocycloalkyl.

For example, each of Q_(x) and Q₂ independently is a bond or methyllinker, and each of T_(x) and T₂ independently is tetrahydropyranyl,piperidinyl substituted by 1, 2, or 3 C₁₋₄ alkyl groups, or cyclohexylsubstituted by N(C₁₋₄ alkyl)₂ wherein one or both of the C₁₋₄ alkyl isoptionally substituted with C₁₋₆ alkoxy;

For example, R⁸⁰⁶ is cyclohexyl substituted by N(C₁₋₄ alkyl)₂ or R⁸⁰⁶ istetrahydropyranyl.

For example, R⁸⁰⁶ is

For example, R⁸⁰¹ is phenyl or 5- or 6-membered heteroaryl substitutedwith O—C₁₋₆ alkyl-R_(x), or R⁸⁰¹ is phenyl substituted withCH₂-tetrahydropyranyl.

For example, in some embodiments, a compound according to some aspectsof the present disclosure is of Formula IVa or IVb:

wherein Z′ is CH or N, and R⁸⁰⁷ is C₂₋₃ alkyl-R_(x).

For example, R⁸⁰⁷ is —CH₂CH₂OH, —CH₂CH₂OCH₃, or —CH₂CH₂OCH₂CH₂OCH₃.

For example, R⁸⁰² is methyl or isopropyl and R⁸⁰³ is methyl or methoxyl.

For example, R⁸⁰⁴ is methyl.

In some embodiments, a compound of the present invention may have thefollowing Formula (V):

or a pharmaceutically acceptable salt or ester thereof.

In this formula:

R₂, R₄ and R₁₂ are each, independently C₁₋₆ alkyl;

R₆ is C₆-C₁₀ aryl or 5- or 6-membered heteroaryl, each of which isoptionally substituted with one or more -Q₂-T₂, wherein Q₂ is a bond orC₁-C₃ alkyl linker optionally substituted with halo, cyano, hydroxyl orC₁-C₆ alkoxy, and T₂ is H, halo, cyano, —OR_(a), —NR_(a)R_(b),—(NR_(a)R_(b)R_(c))⁺A⁻, —C(O)R_(a), —C(O)OR_(a), —C(O)NR_(a)R_(b),—NR_(b)C(O)R_(a), —NR_(b)C(O)OR_(a), —S(O)₂R_(a), —S(O)₂NR_(a)R_(b), orR_(S2), in which each of R_(a), R_(b), and R_(c), independently is H orR_(S3), A⁻ is a pharmaceutically acceptable anion, each of R_(S2) andR_(S3), independently, is C₁-C₆ alkyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4to 12-membered heterocycloalkyl, or 5- or 6-membered heteroaryl, orR_(a) and R_(b), together with the N atom to which they are attached,form a 4 to 12-membered heterocycloalkyl ring having 0 or 1 additionalheteroatom, and each of R_(S2), R_(S3), and the 4 to 12-memberedheterocycloalkyl ring formed by R_(a) and R_(b), is optionallysubstituted with one or more -Q₃-T₃, wherein Q₃ is a bond or C₁-C₃ alkyllinker each optionally substituted with halo, cyano, hydroxyl or C₁-C₆alkoxy, and T₃ is selected from the group consisting of halo, cyano,C₁-C₆ alkyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-memberedheterocycloalkyl, 5- or 6-membered heteroaryl, OR_(d), COOR_(d),—S(O)₂R_(d), —NR_(d)R_(e), and —C(O)NR_(d)R_(e), each of R_(d) and R_(e)independently being H or C₁-C₆ alkyl, or -Q₃-T₃ is oxo; or any twoneighboring -Q₂-T₂, together with the atoms to which they are attachedform a 5- or 6-membered ring optionally containing 1-4 heteroatomsselected from N, O and S and optionally substituted with one or moresubstituents selected from the group consisting of halo, hydroxyl, COOH,C(O)O—C₁-C₆ alkyl, cyano, C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino,di-C₁-C₆ alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-memberedheterocycloalkyl, and 5- or 6-membered heteroaryl;

R₇ is -Q₄-T₄, in which Q₄ is a bond, C₁-C₄ alkyl linker, or C₂-C₄alkenyl linker, each linker optionally substituted with halo, cyano,hydroxyl or C₁-C₆ alkoxy, and T₄ is H, halo, cyano, NR_(f)R_(g),—OR_(f), —C(O)R_(f), —C(O)OR_(f), —C(O)NR_(f)R_(g), —C(O)NR_(f)OR_(g),—NR_(f)C(O)R_(g), —S(O)₂R_(f), or R_(S4), in which each of R_(f) andR_(g), independently is H or R_(S5), each of R_(S4) and R_(S5),independently is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, or 5- or6-membered heteroaryl, and each of R_(S4) and R_(S5) is optionallysubstituted with one or more -Q₅-T₅, wherein Q₅ is a bond, C(O),C(O)NR_(k), NR_(k)C(O), S(O)₂, or C₁-C₃ alkyl linker, R_(k) being H orC₁-C₆ alkyl, and T₅ is H, halo, C₁-C₆ alkyl, hydroxyl, cyano, C₁-C₆alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, 5- or6-membered heteroaryl, or S(O)_(q)R_(q) in which q is 0, 1, or 2 andR_(q) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₅ cycloalkyl,C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, or 5- or 6-memberedheteroaryl, and T₅ is optionally substituted with one or moresubstituents selected from the group consisting of halo, C₁-C₆ alkyl,hydroxyl, cyano, C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-memberedheterocycloalkyl, and 5- or 6-membered heteroaryl except when T₅ is H,halo, hydroxyl, or cyano; or -Q₅-T₅ is oxo; and

R₈ is H, halo, hydroxyl, COOH, cyano, R_(S6), OR_(S6), or COOR_(S6), inwhich R_(S6) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈cycloalkyl, 4 to 12-membered heterocycloalkyl, amino, mono-C₁-C₆alkylamino, or di-C₁-C₆ alkylamino, and R_(S6) is optionally substitutedwith one or more substituents selected from the group consisting ofhalo, hydroxyl, COOH, C(O)O—C₁-C₆ alkyl, cyano, C₁-C₆ alkoxyl, amino,mono-C₁-C₆ alkylamino, and di-C₁-C₆ alkylamino; or R₇ and R₈, togetherwith the N atom to which they are attached, form a 4 to 11-memberedheterocycloalkyl ring having 0 to 2 additional heteroatoms, and the 4 to11-membered heterocycloalkyl ring formed by R₇ and R₈ is optionallysubstituted with one or more -Q₆-T₆, wherein Q₆ is a bond, C(O),C(O)NR_(m), NR_(m)C(O), S(O)₂, or C₁-C₃ alkyl linker, R_(m) being H orC₁-C₆ alkyl, and T₆ is H, halo, C₁-C₆ alkyl, hydroxyl, cyano, C₁-C₆alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, 5- or6-membered heteroaryl, or S(O)_(p)R_(p) in which p is 0, 1, or 2 andR_(p) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl,C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, or 5- or 6-memberedheteroaryl, and T₆ is optionally substituted with one or moresubstituents selected from the group consisting of halo, C₁-C₆ alkyl,hydroxyl, cyano, C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-memberedheterocycloalkyl, and 5- or 6-membered heteroaryl except when T₆ is H,halo, hydroxyl, or cyano; or -Q₆-T₆ is oxo.

For example, R₆ is C₆-C₁₀ aryl or 5- or 6-membered heteroaryl, each ofwhich is optionally, independently substituted with one or more -Q₂-T₂,wherein Q₂ is a bond or C₁-C₃ alkyl linker, and T₂ is H, halo, cyano,—OR_(a), —NR_(a)R_(b), —(NR_(a)R_(b)R_(c))⁺A⁻, —C(O)NR_(a)R_(b),—NR_(b)C(O)R_(a), —S(O)₂R_(a), or R_(S2), in which each of R_(a) andR_(b), independently is H or R_(S3), each of R_(S2) and R_(S3),independently, is C₁-C₆ alkyl, or R_(a) and R_(b), together with the Natom to which they are attached, form a 4 to 7-membered heterocycloalkylring having 0 or 1 additional heteroatom, and each of R_(S2), R_(S3),and the 4 to 7-membered heterocycloalkyl ring formed by R_(a) and R_(b),is optionally, independently substituted with one or more -Q₃-T₃,wherein Q₃ is a bond or C₁-C₃ alkyl linker and T₃ is selected from thegroup consisting of halo, C₁-C₆ alkyl, 4 to 7-membered heterocycloalkyl,OR_(a), —S(O)₂R_(a), and —NR_(d)R_(e), each of R_(d) and R_(e)independently being H or C₁-C₆ alkyl, or -Q₃-T₃ is oxo; or any twoneighboring -Q₂-T₂, together with the atoms to which they are attachedform a 5- or 6-membered ring optionally containing 1-4 heteroatomsselected from N, O and S.

In some embodiments, the compound is of Formula (VI):

or a pharmaceutically acceptable salt thereof, wherein Q₂ is a bond ormethyl linker, T₂ is H, halo, —OR_(a), —NR_(a)R_(b),—(NR_(a)R_(b)R_(c))⁺A⁻, or —S(O)₂NR_(a)R_(b), R₇ is piperidinyl,tetrahydropyran, cyclopentyl, or cyclohexyl, each optionally substitutedwith one -Q₅-T₅ and R₈ is ethyl.

Some aspects of the present disclosure provide the compounds of Formula(VIa):

or a pharmaceutically acceptable salts or esters thereof, wherein R₇,R₈, R_(a), and R_(b) are defined herein.

The compounds of Formula (VIa) can include one or more of the followingfeatures:

For example, each of R_(a) and R_(b) independently is H or C₁-C₆ alkyloptionally substituted with one or more -Q₃-T₃.

For example, one of R_(a) and R_(b) is H.

For example, R_(a) and R_(b), together with the N atom to which they areattached, form a 4 to 7-membered heterocycloalkyl ring having 0 or 1additional heteroatoms to the N atom (e.g., azetidinyl, pyrrolidinyl,imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl,triazolidinyl, piperidinyl, 1,2,3,6-tetrahydropyridinyl, piperazinyl,morpholinyl, 1,4-diazepanyl, 1,4-oxazepanyl,2-oxa-5-azabicyclo[2.2.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl, andthe like) and the ring is optionally substituted with one or more-Q₃-T₃.

For example, R_(a) and R_(b), together with the N atom to which they areattached, form azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl,oxazolidinyl, isoxazolidinyl, triazolidinyl, tetrahydrofuranyl,piperidinyl, 1,2,3,6-tetrahydropyridinyl, piperazinyl, or morpholinyl,and the ring is optionally substituted with one or more -Q₃-T₃.

For example, one or more -Q₃-T₃ are oxo.

For example, Q₃ is a bond or unsubstituted or substituted C₁-C₃ alkyllinker.

For example, T₃ is H, halo, 4 to 7-membered heterocycloalkyl, C₁-C₃alkyl, OR_(a), COOR_(d), —S(O)₂R_(d), or —NR_(d)R_(e).

For example, each of R_(d) and R_(e) independently being H or C₁-C₆alkyl.

For example, R₇ is C₃-C₈ cycloalkyl or 4 to 7-membered heterocycloalkyl,each optionally substituted with one or more -Q₅-T₅.

For example, R₇ is piperidinyl, tetrahydropyran,tetrahydro-2H-thiopyranyl, cyclopentyl, cyclohexyl, pyrrolidinyl, orcycloheptyl, each optionally substituted with one or more -Q₅-T₅.

For example, R₇ is cyclopentyl cyclohexyl or tetrahydro-2H-thiopyranyl,each of which is optionally substituted with one or more -Q₅-T₅.

For example, Q₅ is NHC(O) and T₅ is C₁-C₆ alkyl or C₁-C₆ alkoxy, each

For example, one or more -Q₅-T₅ are oxo.

For example, R₇ is 1-oxide-tetrahydro-2H-thiopyranyl or1,1-dioxide-tetrahydro-2H-thiopyranyl.

For example, Q₅ is a bond and T₅ is amino, mono-C₁-C₆ alkylamino,di-C₁-C₆ alkylamino.

For example, Q₅ is CO, S(O)₂, or NHC(O); and T₅ is C₁-C₆ alkyl, C₁-C₆alkoxyl, C₃-C₈ cycloalkyl, or 4 to 7-membered heterocycloalkyl.

For example, R₈ is H or C₁-C₆ alkyl which is optionally substituted withone or more substituents selected from the group consisting of halo,hydroxyl, COOH, C(O)O—C₁-C₆ alkyl, cyano, C₁-C₆ alkoxyl, amino,mono-C₁-C₆ alkylamino, and di-C₁-C₆ alkylamino.

For example, R₈ is H, methyl, or ethyl.

Other compounds of Formulae (I)-(VIa) suitable for use in thestrategies, treatment modalities, methods, combinations, andcompositions provided herein are described in U.S. Publication20120264734, the contents of which are hereby incorporated by referencein their entireties. The compounds of Formulae (I)-(VIa) are suitablefor administration as part of a combination therapy with one or moreother therapeutic agents, e.g., with an immune checkpoint inhibitor asprovided herein.

In some embodiments of the strategies, treatment modalities, methods,combinations, and compositions provided herein, the EZH2 inhibitor isCompound 44

or a pharmaceutically acceptable salt thereof.

Compound 44 or a pharmaceutically acceptable salt thereof, as describedherein, is potent in targeting both wild type and mutant EZH2. Compound44 is orally bioavailable and has high selectivity to EZH2 compared withother histone methyltransferases (i.e. >20,000 fold selectivity by Ki).Importantly, Compound 44 has target methyl mark inhibition that resultsin the killing of genetically defined cancer cells in vitro. Animalmodels have also shown sustained in vivo efficacy following inhibitionof target methyl mark.

In some embodiments, Compound 44 or a pharmaceutically acceptable saltthereof is administered to the subject at a dose of approximately 100 mgto approximately 3200 mg daily, such as about 100 mg BID to about 1600mg BID (e.g., 100 mg BID, 200 mg BID, 400 mg BID, 800 mg BID, or 1600 mgBID), for treating a germinal center-derived lymphoma.

In some embodiments, Compound 44 or a pharmaceutically acceptable saltthereof is administered to a subject in combination (eithersimultaneously or sequentially) with an immune checkpoint inhibitorprovided herein.

In some embodiments, a compound that can be used in the strategies,treatment modalities, methods, combinations, and compositions presentedhere is:

or stereoisomers thereof or pharmaceutically acceptable salts andsolvates thereof.

In some embodiments, the EZH2 inhibitor may comprise, consistessentially of or consist of GSK-126, having the following formula:

stereoisomers thereof, pharmaceutically acceptable salts or solvatesthereof.

In some embodiments of the strategies, treatment modalities, methods,combinations, and compositions provided herein, the EZH2 inhibitor maycomprise, consist essentially of or consist of

or stereoisomers thereof or pharmaceutically acceptable salts andsolvates thereof.

In certain embodiments, a compound (e.g., EZH2 inhibitor) that can beused in any methods presented here may comprise, consist essentially ofor consist of any of Compounds Ga-Gc:

or a stereoisomer, pharmaceutically acceptable salt, or solvate thereof.

In some embodiments, the EZH2 inhibitor may comprise, consistessentially of or consist of CPI-1205 or GSK343.

In some embodiments of the strategies, treatment modalities, methods,combinations, and compositions provided herein, the EZH2 inhibitor is anEZH2 inhibitor described in U.S. Pat. No. 8,536,179 (describing GSK-126among other compounds and corresponding to WO 2011/140324), the entirecontents of each of which are incorporated herein by reference.

In some embodiments of the strategies, treatment modalities, methods,combinations, and compositions provided herein, the EZH2 inhibitor is anEZH2 inhibitor described in PCT/US2014/015706, published asWO/2014/124418, in PCT/US2013/025639, published as WO/2013/120104, andin U.S. Ser. No. 14/839,273, published as US 2015/0368229, the entirecontents of each of which are incorporated herein by reference. In someembodiments of the strategies, treatment modalities, methods,combinations, and compositions provided herein, the EZH2 inhibitor is acompound of the formula:

or a pharmaceutically acceptable salt thereof (see, for example US2015/0368229, the contents of which are incorporated herein).

In some embodiments, the EZH2 inhibitor is a small molecule that is usedas the compound itself, i.e., as the free base or “naked” molecule. Insome embodiments, the EZH2 inhibitor is a salt thereof, e.g., a mono-HClor tri-HCl salt, mono-HBr or tri-HBr salt of the naked molecule.

Representative compounds that are suitable for the strategies, treatmentmodalities, methods, combinations, and compositions provided hereininclude compounds listed in Table 1.

In the table below, each occurrence of

should be construed as

TABLE 1 Compound Number Structure MS (M + 1)⁺   1

501.39   2

543.22   3

486.21   4

529.30  11

558.45  12

559.35  13

517.3  14

557.4  16

515.4  20

614.4  21

614.4  27

516.35  36

557.35  39

572.35  40

572.35  42

572.4  43

572.6  44

573.40  47

530.35  59

587.40  60

601.30  61

599.35  62

601.35  63

613.35  65

531.30  66

586.40  67

585.25  68

585.35  69

557.25  70

573.40  71

573.40  72

575.35  73

572.10  74

575.35  75

571.25  76

587.40  77

587.45  78

587.20  79

589.35  80

589.30  81

607.35  82

543.40  83

559.80  84

561.25  85

 86

585.37  87

600.30  88

587.40  89

503.40  90

517.30  91

531.35  92

545.40  93

557.35  94

559.20  95

599.35 (M + Na)  96

577.25  97

571.40  98

547.35  99

561.30 100

591.25 101

546.35 102

560.20 103

567.30 104

585.25 105

585.40 107

108

530.35 114

573.25 115

642.45 116

545.15 117

489.20 119

609.35 122

587.55 124

650.85 125

614.75 126

572.35 127

656.65 128

586.45 129

628.35 130

591.2 131

587.35 132

589.25 133

605.25 135

621.40 136

621.45 137

589.35 138

627.5 141

614.65 142

603.45 143

578.35 144

609.15 146

641.50 178

593.60

As used herein, “alkyl”, “C₁, C₂, C₃, C₄, CS or C₆ alkyl” or “C₁-C₆alkyl” is intended to include C₁, C₂, C₃, C₄, C₅ or C₆ straight chain(linear) saturated aliphatic hydrocarbon groups and C₃, C₄, C₅ or C₆branched saturated aliphatic hydrocarbon groups. For example, C₁-C₆alkyl is intended to include C₁, C₂, C₃, C₄, C₅ and C₆ alkyl groups.Examples of alkyl include, moieties having from one to six carbon atoms,such as, but not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl,s-butyl, t-butyl, n-pentyl, s-pentyl or n-hexyl.

In certain embodiments, a straight chain or branched alkyl has six orfewer carbon atoms (e.g., C₁-C₆ for straight chain, C₃-C₆ for branchedchain), and in some embodiments, a straight chain or branched alkyl hasfour or fewer carbon atoms.

As used herein, the term “cycloalkyl” refers to a saturated orunsaturated nonaromatic hydrocarbon mono- or multi-ring (e.g., fused,bridged, or spiro rings) system having 3 to 30 carbon atoms (e.g.,C₃-C₁₀). Examples of cycloalkyl include, but are not limited to,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, and adamantyl.The term “heterocycloalkyl” refers to a saturated or unsaturatednonaromatic 3-8 membered monocyclic, 7-12 membered bicyclic (fused,bridged, or spiro rings), or 11-14 membered tricyclic ring system(fused, bridged, or spiro rings) having one or more heteroatoms (such asO, N, S, or Se), unless specified otherwise. Examples ofheterocycloalkyl groups include, but are not limited to, piperidinyl,piperazinyl, pyrrolidinyl, dioxanyl, tetrahydrofuranyl, isoindolinyl,indolinyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, isoxazolidinyl,triazolidinyl, tetrahydrofuranyl, oxiranyl, azetidinyl, oxetanyl,thietanyl, 1,2,3,6-tetrahydropyridinyl, tetrahydropyranyl,dihydropyranyl, pyranyl, morpholinyl, 1,4-diazepanyl, 1,4-oxazepanyl,2-oxa-5-azabicyclo[2.2.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl,2-oxa-6-azaspiro[3.3]heptanyl, 2,6-diazaspiro[3.3]heptanyl,1,4-dioxa-8-azaspiro[4.5]decanyl and the like.

The term “optionally substituted alkyl” refers to unsubstituted alkyl oralkyl having designated substituents replacing one or more hydrogenatoms on one or more carbons of the hydrocarbon backbone. Suchsubstituents can include, for example, alkyl, alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino(including alkylamino, dialkylamino, arylamino, diarylamino andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

An “arylalkyl” or an “aralkyl” moiety is an alkyl substituted with anaryl (e.g., phenylmethyl (benzyl)). An “alkylaryl” moiety is an arylsubstituted with an alkyl (e.g., methylphenyl).

As used herein, “alkyl linker” is intended to include C₁, C₂, C₃, C₄, C₅or C₆ straight chain (linear) saturated divalent aliphatic hydrocarbongroups and C₃, C₄, C₅ or C₆ branched saturated aliphatic hydrocarbongroups. For example, C₁-C₆ alkyl linker is intended to include C₁, C₂,C₃, C₄, C₅ and C₆ alkyl linker groups. Examples of alkyl linker include,moieties having from one to six carbon atoms, such as, but not limitedto, methyl (—CH₂—), ethyl (—CH₂CH₂—), n-propyl (—CH₂CH₂CH₂—), i-propyl(—CHCH₃CH₂—), n-butyl (—CH₂CH₂CH₂CH₂—), s-butyl (—CHCH₃CH₂CH₂—), i-butyl(—C(CH₃)₂CH₂—), n-pentyl (—CH₂CH₂CH₂CH₂CH₂—), s-pentyl(—CHCH₃CH₂CH₂CH₂—) or n-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₂—).

“Alkenyl” includes unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but that contain atleast one double bond. For example, the term “alkenyl” includes straightchain alkenyl groups (e.g., ethenyl, propenyl, butenyl, pentenyl,hexenyl, heptenyl, octenyl, nonenyl, decenyl), and branched alkenylgroups. In certain embodiments, a straight chain or branched alkenylgroup has six or fewer carbon atoms in its backbone (e.g., C₂-C₆ forstraight chain, C₃-C₆ for branched chain). The term “C₂-C₆” includesalkenyl groups containing two to six carbon atoms. The term “C₃-C₆”includes alkenyl groups containing three to six carbon atoms.

The term “optionally substituted alkenyl” refers to unsubstitutedalkenyl or alkenyl having designated substituents replacing one or morehydrogen atoms on one or more hydrocarbon backbone carbon atoms. Suchsubstituents can include, for example, alkyl, alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino(including alkylamino, dialkylamino, arylamino, diarylamino andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

“Alkynyl” includes unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but which containat least one triple bond. For example, “alkynyl” includes straight chainalkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl,heptynyl, octynyl, nonynyl, decynyl), and branched alkynyl groups. Incertain embodiments, a straight chain or branched alkynyl group has sixor fewer carbon atoms in its backbone (e.g., C₂-C₆ for straight chain,C₃-C₆ for branched chain). The term “C₂-C₆” includes alkynyl groupscontaining two to six carbon atoms. The term “C₃-C₆” includes alkynylgroups containing three to six carbon atoms.

The term “optionally substituted alkynyl” refers to unsubstitutedalkynyl or alkynyl having designated substituents replacing one or morehydrogen atoms on one or more hydrocarbon backbone carbon atoms. Suchsubstituents can include, for example, alkyl, alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino(including alkylamino, dialkylamino, arylamino, diarylamino andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

Other optionally substituted moieties (such as optionally substitutedcycloalkyl, heterocycloalkyl, aryl, or heteroaryl) include both theunsubstituted moieties and the moieties having one or more of thedesignated substituents. For example, substituted heterocycloalkylincludes those substituted with one or more alkyl groups, such as2,2,6,6-tetramethyl-piperidinyl and2,2,6,6-tetramethyl-1,2,3,6-tetrahydropyridinyl.

“Aryl” includes groups with aromaticity, including “conjugated,” ormulticyclic systems with at least one aromatic ring and do not containany heteroatom in the ring structure. Examples include phenyl, benzyl,1,2,3,4-tetrahydronaphthalenyl, etc.

“Heteroaryl” groups are aryl groups, as defined above, except havingfrom one to four heteroatoms in the ring structure, and may also bereferred to as “aryl heterocycles” or “heteroaromatics.” As used herein,the term “heteroaryl” is intended to include a stable 5-, 6-, or7-membered monocyclic or 7-, 8-, 9-, 10-, 11- or 12-membered bicyclicaromatic heterocyclic ring which consists of carbon atoms and one ormore heteroatoms, e.g., 1 or 1-2 or 1-3 or 1-4 or 1-5 or 1-6heteroatoms, or e.g., 1, 2, 3, 4, 5, or 6 heteroatoms, independentlyselected from the group consisting of nitrogen, oxygen and sulfur. Thenitrogen atom may be substituted or unsubstituted (i.e., N or NR whereinR is H or other substituents, as defined). The nitrogen and sulfurheteroatoms may optionally be oxidized (i.e., N→O and S(O)_(p), wherep=1or 2). It is to be noted that total number of S and O atoms in thearomatic heterocycle is not more than 1.

Examples of heteroaryl groups include pyrrole, furan, thiophene,thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole,oxazole, isoxazole, pyridine, pyrazine, pyridazine, pyrimidine, and thelike.

Furthermore, the terms “aryl” and “heteroaryl” include multicyclic aryland heteroaryl groups, e.g., tricyclic, bicyclic, e.g., naphthalene,benzoxazole, benzodioxazole, benzothiazole, benzoimidazole,benzothiophene, methylenedioxyphenyl, quinoline, isoquinoline,naphthrydine, indole, benzofuran, purine, benzofuran, deazapurine,indolizine.

In the case of multicyclic aromatic rings, only one of the rings needsto be aromatic (e.g., 2,3-dihydroindole), although all of the rings maybe aromatic (e.g., quinoline). The second ring can also be fused orbridged.

The cycloalkyl, heterocycloalkyl, aryl, or heteroaryl ring can besubstituted at one or more ring positions (e.g., the ring-forming carbonor heteroatom such as N) with such substituents as described above, forexample, alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkoxy,alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl,aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl,aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylthiocarbonyl, phosphate, phosphonato, phosphinato, amino (includingalkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety. Aryl and heteroarylgroups can also be fused or bridged with alicyclic or heterocyclicrings, which are not aromatic so as to form a multicyclic system (e.g.,tetralin, methylenedioxyphenyl).

As used herein, “carbocycle” or “carbocyclic ring” is intended toinclude any stable monocyclic, bicyclic or tricyclic ring having thespecified number of carbons, any of which may be saturated, unsaturated,or aromatic. Carbocycle includes cycloalkyl and aryl. For example, aC₃-C₁₄ carbocycle is intended to include a monocyclic, bicyclic ortricyclic ring having 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 carbonatoms. Examples of carbocycles include, but are not limited to,cyclopropyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl,cyclohexyl, cycloheptenyl, cycloheptyl, cycloheptenyl, adamantyl,cyclooctyl, cyclooctenyl, cyclooctadienyl, fluorenyl, phenyl, naphthyl,indanyl, adamantyl and tetrahydronaphthyl. Bridged rings are alsoincluded in the definition of carbocycle, including, for example,[3.3.0]bicyclooctane, [4.3.0]bicyclononane, [4.4.0]bicyclodecane and[2.2.2]bicyclooctane. A bridged ring occurs when one or more carbonatoms link two non-adjacent carbon atoms. In some embodiments, bridgerings are one or two carbon atoms. It is noted that a bridge alwaysconverts a monocyclic ring into a tricyclic ring. When a ring isbridged, the substituents recited for the ring may also be present onthe bridge. Fused (e.g., naphthyl, tetrahydronaphthyl) and spiro ringsare also included.

As used herein, “heterocycle” or “heterocyclic group” includes any ringstructure (saturated, unsaturated, or aromatic) which contains at leastone ring heteroatom (e.g., N, O or S). Heterocycle includesheterocycloalkyl and heteroaryl. Examples of heterocycles include, butare not limited to, morpholine, pyrrolidine, tetrahydrothiophene,piperidine, piperazine, oxetane, pyran, tetrahydropyran, azetidine, andtetrahydrofuran.

Examples of heterocyclic groups include, but are not limited to,acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl,benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl,benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl,benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl,chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl,isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl,isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl,naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl,1,2,4-oxadiazol5(4H)-one, oxazolidinyl, oxazolyl, oxindolyl,pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl,phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl,piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl,pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl,pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl,pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl,quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl,1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl,thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl,thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl,1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl and xanthenyl.

The term “substituted,” as used herein, means that any one or morehydrogen atoms on the designated atom is replaced with a selection fromthe indicated groups, provided that the designated atom's normal valencyis not exceeded, and that the substitution results in a stable compound.When a substituent is oxo or keto (i.e., ═O), then 2 hydrogen atoms onthe atom are replaced. Keto substituents are not present on aromaticmoieties. Ring double bonds, as used herein, are double bonds that areformed between two adjacent ring atoms (e.g., C═C, C═N or N═N). “Stablecompound” and “stable structure” are meant to indicate a compound thatis sufficiently robust to survive isolation to a useful degree of purityfrom a reaction mixture, and formulation into an efficacious therapeuticagent.

When a bond to a substituent is shown to cross a bond connecting twoatoms in a ring, then such substituent may be bonded to any atom in thering. When a substituent is listed without indicating the atom via whichsuch substituent is bonded to the rest of the compound of a givenformula, then such substituent may be bonded via any atom in suchformula. Combinations of substituents and/or variables are permissible,but only if such combinations result in stable compounds.

When any variable (e.g., R₁) occurs more than one time in anyconstituent or formula for a compound, its definition at each occurrenceis independent of its definition at every other occurrence. Thus, forexample, if a group is shown to be substituted with 0-2 R₁ moieties,then the group may optionally be substituted with up to two R₁ moietiesand R₁ at each occurrence is selected independently from the definitionof R₁. Also, combinations of substituents and/or variables arepermissible, but only if such combinations result in stable compounds.

The term “hydroxy” or “hydroxyl” includes groups with an —OH or —O.

As used herein, “halo” or “halogen” refers to fluoro, chloro, bromo andiodo. The term “perhalogenated” generally refers to a moiety wherein allhydrogen atoms are replaced by halogen atoms. The term “haloalkyl” or“haloalkoxyl” refers to an alkyl or alkoxyl substituted with one or morehalogen atoms.

The term “carbonyl” includes compounds and moieties which contain acarbon connected with a double bond to an oxygen atom. Examples ofmoieties containing a carbonyl include, but are not limited to,aldehydes, ketones, carboxylic acids, amides, esters, anhydrides, etc.

The term “carboxyl” refers to —COOH or its C₁-C₆ alkyl ester.

“Acyl” includes moieties that contain the acyl radical (R—C(O)—) or acarbonyl group. “Substituted acyl” includes acyl groups where one ormore of the hydrogen atoms are replaced by, for example, alkyl groups,alkynyl groups, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, amino (including alkylamino, dialkylamino,arylamino, diarylamino and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

“Aroyl” includes moieties with an aryl or heteroaromatic moiety bound toa carbonyl group. Examples of aroyl groups include phenylcarboxy,naphthyl carboxy, etc.

“Alkoxyalkyl,” “alkylaminoalkyl,” and “thioalkoxyalkyl” include alkylgroups, as described above, wherein oxygen, nitrogen, or sulfur atomsreplace one or more hydrocarbon backbone carbon atoms.

The term “alkoxy” or “alkoxyl” includes substituted and unsubstitutedalkyl, alkenyl and alkynyl groups covalently linked to an oxygen atom.Examples of alkoxy groups or alkoxyl radicals include, but are notlimited to, methoxy, ethoxy, isopropyloxy, propoxy, butoxy and pentoxygroups. Examples of substituted alkoxy groups include halogenated alkoxygroups. The alkoxy groups can be substituted with groups such asalkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, amino (including alkylamino, dialkylamino,arylamino, diarylamino, and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moieties. Examples of halogen substituted alkoxygroups include, but are not limited to, fluoromethoxy, difluoromethoxy,trifluoromethoxy, chloromethoxy, dichloromethoxy and trichloromethoxy.

The term “ether” or “alkoxy” includes compounds or moieties whichcontain an oxygen bonded to two carbon atoms or heteroatoms. Forexample, the term includes “alkoxyalkyl,” which refers to an alkyl,alkenyl, or alkynyl group covalently bonded to an oxygen atom which iscovalently bonded to an alkyl group.

The term “ester” includes compounds or moieties which contain a carbonor a heteroatom bound to an oxygen atom which is bonded to the carbon ofa carbonyl group. The term “ester” includes alkoxycarboxy groups such asmethoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl,pentoxycarbonyl, etc.

The term “thioalkyl” includes compounds or moieties which contain analkyl group connected with a sulfur atom. The thioalkyl groups can besubstituted with groups such as alkyl, alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, carboxyacid, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, amino (includingalkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moieties.

The term “thiocarbonyl” or “thiocarboxy” includes compounds and moietieswhich contain a carbon connected with a double bond to a sulfur atom.

The term “thioether” includes moieties which contain a sulfur atombonded to two carbon atoms or heteroatoms. Examples of thioethersinclude, but are not limited to alkthioalkyls, alkthioalkenyls, andalkthioalkynyls. The term “alkthioalkyls” include moieties with analkyl, alkenyl, or alkynyl group bonded to a sulfur atom which is bondedto an alkyl group. Similarly, the term “alkthioalkenyls” refers tomoieties wherein an alkyl, alkenyl or alkynyl group is bonded to asulfur atom which is covalently bonded to an alkenyl group; andalkthioalkynyls” refers to moieties wherein an alkyl, alkenyl or alkynylgroup is bonded to a sulfur atom which is covalently bonded to analkynyl group.

As used herein, “amine” or “amino” refers to unsubstituted orsubstituted —NH₂. “Alkylamino” includes groups of compounds whereinnitrogen of —NH₂ is bound to at least one alkyl group. Examples ofalkylamino groups include benzylamino, methylamino, ethylamino,phenethylamino, etc. “Dialkylamino” includes groups wherein the nitrogenof —NH₂ is bound to at least two additional alkyl groups. Examples ofdialkylamino groups include, but are not limited to, dimethylamino anddiethylamino. “Arylamino” and “diarylamino” include groups wherein thenitrogen is bound to at least one or two aryl groups, respectively.“Aminoaryl” and “aminoaryloxy” refer to aryl and aryloxy substitutedwith amino. “Alkylarylamino,” “alkylaminoaryl” or “arylaminoalkyl”refers to an amino group which is bound to at least one alkyl group andat least one aryl group. “Alkaminoalkyl” refers to an alkyl, alkenyl, oralkynyl group bound to a nitrogen atom which is also bound to an alkylgroup. “Acylamino” includes groups wherein nitrogen is bound to an acylgroup. Examples of acylamino include, but are not limited to,alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido groups.

The term “amide” or “aminocarboxy” includes compounds or moieties thatcontain a nitrogen atom that is bound to the carbon of a carbonyl or athiocarbonyl group. The term includes “alkaminocarboxy” groups thatinclude alkyl, alkenyl or alkynyl groups bound to an amino group whichis bound to the carbon of a carbonyl or thiocarbonyl group. It alsoincludes “arylaminocarboxy” groups that include aryl or heteroarylmoieties bound to an amino group that is bound to the carbon of acarbonyl or thiocarbonyl group. The terms “alkylaminocarboxy”,“alkenylaminocarboxy”, “alkynylaminocarboxy” and “arylaminocarboxy”include moieties wherein alkyl, alkenyl, alkynyl and aryl moieties,respectively, are bound to a nitrogen atom which is in turn bound to thecarbon of a carbonyl group. Amides can be substituted with substituentssuch as straight chain alkyl, branched alkyl, cycloalkyl, aryl,heteroaryl or heterocycle. Substituents on amide groups may be furthersubstituted.

Compounds of the present invention that contain nitrogens can beconverted to N-oxides by treatment with an oxidizing agent (e.g.,3-chloroperoxybenzoic acid (mCPBA) and/or hydrogen peroxides) to affordother compounds of the present invention. Thus, all shown and claimednitrogen-containing compounds are considered, when allowed by valencyand structure, to include both the compound as shown and its N-oxidederivative (which can be designated as N→O or N⁺—O—). Furthermore, inother instances, the nitrogens in the compounds of the present inventioncan be converted to N-hydroxy or N-alkoxy compounds. For example,N-hydroxy compounds can be prepared by oxidation of the parent amine byan oxidizing agent such as m-CPBA. All shown and claimednitrogen-containing compounds are also considered, when allowed byvalency and structure, to cover both the compound as shown and itsN-hydroxy (i.e., N—OH) and N-alkoxy (i.e., N—OR, wherein R issubstituted or unsubstituted C₁-C₆ alkyl, C₁-C₆ alkenyl, C₁-C₆ alkynyl,3-14-membered carbocycle or 3-14-membered heterocycle) derivatives.

“Isomerism” means compounds that have identical molecular formulae butdiffer in the sequence of bonding of their atoms or in the arrangementof their atoms in space. Isomers that differ in the arrangement of theiratoms in space are termed “stereoisomers.” Stereoisomers that are notmirror images of one another are termed “diastereoisomers,” andstereoisomers that are non-superimposable mirror images of each otherare termed “enantiomers” or sometimes optical isomers. A mixturecontaining equal amounts of individual enantiomeric forms of oppositechirality is termed a “racemic mixture.”

A carbon atom bonded to four nonidentical substituents is termed a“chiral center.”

“Chiral isomer” means a compound with at least one chiral center.Compounds with more than one chiral center may exist either as anindividual diastereomer or as a mixture of diastereomers, termed“diastereomeric mixture.” When one chiral center is present, astereoisomer may be characterized by the absolute configuration (R or S)of that chiral center. Absolute configuration refers to the arrangementin space of the substituents attached to the chiral center. Thesubstituents attached to the chiral center under consideration areranked in accordance with the Sequence Rule of Cahn, Ingold and Prelog.(Cahn et al., Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahnet al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J. Chem. Soc. 1951(London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J. Chem.Educ. 1964, 41, 116).

“Geometric isomer” means the diastereomers that owe their existence tohindered rotation about double bonds or a cycloalkyl linker (e.g.,1,3-cyclobutyl). These configurations are differentiated in their namesby the prefixes cis and trans, or Z and E, which indicate that thegroups are on the same or opposite side of the double bond in themolecule according to the Cahn-Ingold-Prelog rules.

It is to be understood that the small molecule EZH2 inhibitors providedherein may be depicted as different chiral isomers or geometric isomers.It should also be understood that when compounds have chiral isomeric orgeometric isomeric forms, all isomeric forms are intended to be includedin the scope of the present invention, and the naming of the compoundsdoes not exclude any isomeric forms.

Furthermore, the structures and other compounds discussed in thisinvention include all atropic isomers thereof. “Atropic isomers” are atype of stereoisomer in which the atoms of two isomers are arrangeddifferently in space. Atropic isomers owe their existence to arestricted rotation caused by hindrance of rotation of large groupsabout a central bond. Such atropic isomers typically exist as a mixture,however as a result of recent advances in chromatography techniques, ithas been possible to separate mixtures of two atropic isomers in selectcases.

“Tautomer” is one of two or more structural isomers that exist inequilibrium and is readily converted from one isomeric form to another.This conversion results in the formal migration of a hydrogen atomaccompanied by a switch of adjacent conjugated double bonds. Tautomersexist as a mixture of a tautomeric set in solution. In solutions wheretautomerization is possible, a chemical equilibrium of the tautomerswill be reached. The exact ratio of the tautomers depends on severalfactors, including temperature, solvent and pH. The concept of tautomersthat are interconvertible by tautomerization is called tautomerism.

Of the various types of tautomerism that are possible, two are commonlyobserved. In keto-enol tautomerism a simultaneous shift of electrons anda hydrogen atom occurs. Ring-chain tautomerism arises as a result of thealdehyde group (—CHO) in a sugar chain molecule reacting with one of thehydroxy groups (—OH) in the same molecule to give it a cyclic(ring-shaped) form as exhibited by glucose.

Common tautomeric pairs are: ketone-enol, amide-nitrile, lactam-lactim,amide-imidic acid tautomerism in heterocyclic rings (e.g., innucleobases such as guanine, thymine and cytosine), imine-enamine andenamine-enamine. An example of keto-enol equilibria is betweenpyridin-2(1H)-ones and the corresponding pyridin-2-ols, as shown below.

It is to be understood that the compounds of the present invention maybe depicted as different tautomers. It should also be understood thatwhen compounds have tautomeric forms, all tautomeric forms are intendedto be included in the scope of the present invention, and the naming ofthe compounds does not exclude any tautomer form.

The EZH2 inhibitors of Formulae (I)-(VIa) disclosed herein include thecompounds themselves, as well as their salts and their solvates, ifapplicable. A salt, for example, can be formed between an anion and apositively charged group (e.g., amino) on an aryl- orheteroaryl-substituted benzene compound. Suitable anions includechloride, bromide, iodide, sulfate, bisulfate, sulfamate, nitrate,phosphate, citrate, methanesulfonate, trifluoroacetate, glutamate,glucuronate, glutarate, malate, maleate, succinate, fumarate, tartrate,tosylate, salicylate, lactate, naphthalenesulfonate, and acetate (e.g.,trifluoroacetate). The term “pharmaceutically acceptable anion” refersto an anion suitable for forming a pharmaceutically acceptable salt.Likewise, a salt can also be formed between a cation and a negativelycharged group (e.g., carboxylate) on an aryl- or heteroaryl-substitutedbenzene compound. Suitable cations include sodium ion, potassium ion,magnesium ion, calcium ion, and an ammonium cation such astetramethylammonium ion. The aryl- or heteroaryl-substituted benzenecompounds also include those salts containing quaternary nitrogen atoms.In the salt form, it is understood that the ratio of the compound to thecation or anion of the salt can be 1:1, or any ration other than 1:1,e.g., 3:1, 2:1, 1:2, or 1:3.

Additionally, the EZH2 inhibitory compounds of the present invention,for example, the salts of the compounds, can exist in either hydrated orunhydrated (the anhydrous) form or as solvates with other solventmolecules. Nonlimiting examples of hydrates include monohydrates,dihydrates, etc. Nonlimiting examples of solvates include ethanolsolvates, acetone solvates, etc.

“Solvate” means solvent addition forms that contain eitherstoichiometric or non-stoichiometric amounts of solvent. Some compoundshave a tendency to trap a fixed molar ratio of solvent molecules in thecrystalline solid state, thus forming a solvate. If the solvent is waterthe solvate formed is a hydrate; and if the solvent is alcohol, thesolvate formed is an alcoholate. Hydrates are formed by the combinationof one or more molecules of water with one molecule of the substance inwhich the water retains its molecular state as H₂O.

As used herein, the term “analog” refers to a chemical compound that isstructurally similar to another but differs slightly in composition (asin the replacement of one atom by an atom of a different element or inthe presence of a particular functional group, or the replacement of onefunctional group by another functional group). Thus, an analog is acompound that is similar or comparable in function and appearance, butnot in structure or origin to the reference compound.

As used herein, the term “derivative” refers to compounds that have acommon core structure, and are substituted with various groups asdescribed herein. For example, all of the compounds represented byFormula (I) are aryl- or heteroaryl-substituted benzene compounds, andhave Formula (I) as a common core.

Some embodiments of the present disclosure embrace some or all isotopesof atoms occurring in the present EZH2 inhibitory compounds. Isotopesinclude those atoms having the same atomic number but different massnumbers. By way of general example and without limitation, isotopes ofhydrogen include tritium and deuterium, and isotopes of carbon includeC-13 and C-14.

In certain aspects of the disclosure an inhibitor of EZH2 “selectivelyinhibits” histone methyltransferase activity of the mutant EZH2 when itinhibits histone methyltransferase activity of the mutant EZH2 moreeffectively than it inhibits histone methyltransferase activity ofwild-type EZH2. For example, in some embodiments the selective inhibitorhas an IC50 for the mutant EZH2 that is at least 40 percent lower thanthe IC50 for wild-type EZH2. In some embodiments, the selectiveinhibitor has an IC50 for the mutant EZH2 that is at least 50 percentlower than the IC50 for wild-type EZH2. In some embodiments, theselective inhibitor has an IC50 for the mutant EZH2 that is at least 60percent lower than the IC50 for wild-type EZH2. In some embodiments, theselective inhibitor has an IC50 for the mutant EZH2 that is at least 70percent lower than the IC50 for wild-type EZH2. In some embodiments, theselective inhibitor has an IC50 for the mutant EZH2 that is at least 80percent lower than the IC50 for wild-type EZH2. In some embodiments, theselective inhibitor has an IC50 for the mutant EZH2 that is at least 90percent lower than the IC50 for wild-type EZH2.

In some embodiments, the selective inhibitor of a mutant EZH2 exertsessentially no inhibitory effect on wild-type EZH2.

In some embodiments, an inhibitor of the disclosure inhibits conversionof H3-K27me2 to H3-K27me3. In some embodiments the inhibitor is said toinhibit trimethylation of H3-K27. Since conversion of H3-K27me1 toH3-K27me2 precedes conversion of H3-K27me2 to H3-K27me3, an inhibitor ofconversion of H3-K27me1 to H3-K27me2 naturally also inhibits conversionof H3-K27me2 to H3-K27me3, i.e., it inhibits trimethylation of H3-K27.It is also possible to inhibit conversion of H3-K27me2 to H3-K27me3without inhibition of conversion of H3-K27me1 to H3-K27me2. Inhibitionof this type would also result in inhibition of trimethylation ofH3-K27, albeit without inhibition of dimethylation of H3-K27.

In some embodiments an inhibitor of the disclosure inhibits conversionof H3-K27me1 to H3-K27me2 and the conversion of H3-K27me2 to H3-K27me3.Such inhibitor may directly inhibit the conversion of H3-K27me1 toH3-K27me2 alone. Alternatively, such inhibitor may directly inhibit boththe conversion of H3-K27me1 to H3-K27me2 and the conversion of H3-K27me2to H3-K27me3.

In certain aspects of the invention, an EZH2 inhibitor of the disclosureinhibits histone methyltransferase activity. Inhibition of histonemethyltransferase activity can be detected using any suitable method.The inhibition can be measured, for example, either in terms of rate ofhistone methyltransferase activity or as product of histonemethyltransferase activity.

In some embodiments, strategies, treatment modalities, methods,combinations, and compositions are provided that are characterized by ameasurable inhibition of EZH2 activity, for example, a measureable EZH2inhibition as compared to a suitable control. In some embodiments, EZH2inhibition is at least 10 percent inhibition compared to a suitablecontrol, e.g., an EZH2 activity observed or expected in an untreatedcontrol cell, tissue, or subject. In some embodiments, the rate of EZH2enzymatic activity in the presence of the EZH2 inhibitor is less than orequal to 90 percent of the corresponding enzymatic activity in theabsence of the EZH2 inhibitor. In some embodiments, EZH2 inhibition inthe presence of the EZH2 inhibitor is at least 20, 25, 30, 40, 50, 60,70, 75, 80, 90, or 95 percent inhibition as compared to a suitablecontrol, e.g., to activity in the absence of the inhibitor. In someembodiments, inhibition is at least 99 percent inhibition compared to asuitable control. That is, the rate of enzymatic activity in thepresence of the inhibitor is less than or equal to 1 percent of thecorresponding activity in the absence of the inhibitor.

Immune Checkpoint Inhibitors

Immune checkpoint proteins inhibit the action of the immune cells (e.g.,T cells) against certain cells. Immune checkpoint signaling plays animportant role in balancing a subject's immune response against cellstargeted by the immune system (e.g., infected or malignant cells), andcells that are not targeted by immune system effectors (e.g., healthycells). Without wishing to be bound by any particular theory, it isbelieved that evasion of some cancer cells from immune systemsurveillance and destruction is mediated by aberrant immune checkpointsignaling, wherein cancer cells modulate or abolish the host's immuneresponse by activating one or more immune checkpoint signaling pathwaysin the host's immune cells.

Various immune checkpoint signaling proteins have been identified, forexample, and without limitation, CTLA4, PD-1, PD-L1, LAG3, B7-H3, andTim3, and immune checkpoint inhibitors targeting such immune checkpointproteins have been developed. Such immune checkpoint inhibitors decreaseor abolish the activity of the immune checkpoint signaling pathway theytarget and can thus boost the subject's immune response, e.g., againstpathologic cells that otherwise escape proper immune systemsurveillance. For example, some immune checkpoint inhibitors have beenreported to effectively inhibit immune checkpoint signaling thatprevented a T-cell mediated attack of an infected or cancerous cell.Accordingly, the immune checkpoint inhibitors described herein enable orsupport immune system surveillance and effector functions (e.g., in theform of a T-cell attack) targeted at malignant or infective cells. Someof the immune checkpoint inhibitors referred to herein includemonoclonal antibodies that specifically bind and inhibit an activity ofone or more checkpoint protein(s) on an immune cell (e.g. a T cell).Immune checkpoint inhibitors of the disclosure may be used to boost thesubject's immune response against any type of cancer cell.

While any checkpoint protein may be targeted, exemplary immunecheckpoint inhibitors of the disclosure may target, bind, and/or inhibitan activity of a protein including, but not limited to, CTLA4, PD-1,PD-L1, LAG3, B7-H3, Tim3 or any combination thereof. Immune checkpointinhibitors that target, bind, and/or inhibit an activity of CTLA4 maycomprise Ipilimumab, Ticilimumab, AGEN-1884 or a combination thereof.Immune checkpoint inhibitors that target, bind, and/or inhibit anactivity of PD-1 and/or PD-L1 may comprise Nivolumab, Pembrolizumab,Atezolizumab, Durvalumab, Avelumab, BMS-936559, AMP-224, MEDI-0680,TSR-042, BGB-108, STI-1014, KY-1003, ALN-PDL, BGB-A317, KD-033,REGN-2810, PDR-001, SHR-1210, MGD-013, PF-06801591, CX-072 or acombination thereof. Immune checkpoint inhibitors that target, bind,and/or inhibit an activity of LAG3 may comprise IMP-731, LAG-525,BMS-986016, GSK-2831781 or a combination thereof. Immune checkpointinhibitors that target, bind, and/or inhibit an activity of B7-H3 maycomprise Enoblituzumab, 1241-8H9, DS-5573 or a combination thereof.Immune checkpoint inhibitors that target, bind, and/or inhibit anactivity of Tim3 may comprise MBG-453.

In some embodiments, the immune checkpoint inhibitor is PD-L1. Someaspects of the disclosure provide methods comprising detectingexpression of PD-L1 in a subject having cancer. In some embodiments, thesubject has been administered an effective amount of an anti-cancerdrug, e.g., of an enhancer of zeste homolog 2 (EZH2) inhibitor. Someaspects of the disclosure relate to methods comprising administering toa subject having a cancer expressing PD-L1, e.g., a cancer showingexpression and/or upregulation of PD-L1 after treatment with ananti-cancer drug, e.g., after treatment with an EZH2 inhibitor, aneffective amount of a PD-L1 inhibitor, either alone or in combinationwith administration of an anti-cancer drug, e.g., an EZH2 inhibitor.

Genomic sequences and transcripts encoding PD-L1 as well as PD-L1proteins are known to those of ordinary skill in the art. ExemplaryPD-L1 sequences are described herein and additional suitable PD-L1sequences will be apparent to those of skill in the art based on thepresent disclosure. Exemplary suitable PD-L1 sequences include, withoutlimitation: (GenBank Accession No. NP_054862.1) programmed cell death 1ligand 1 isoform a precursor [Homo sapiens]

(SEQ ID No: 1) MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET

(GenBank Accession No. NP_001254635.1) programmed cell death 1 ligand 1isoform b precursor [Homo sapiens]

(SEQ ID No: 2) MRIFAVFIFMTYWHLLNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET 

(GenBank Accession No. NP_001300958.1) programmed cell death 1 ligand 1isoform c precursor [Homo sapiens]

(SEQ ID No: 3) MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPGNILNVSIKICLTLSPST 

Additional suitable PD-L1 sequences, as well as methods for thedetection and quantification of PD-L1 proteins and transcripts in targetcells, e.g., cancer cells, will be apparent to the skilled artisan. Suchdetection methods include, without limitation, protein detectionmethods, such as immunohistochemistry and western blot, RNA detectionmethods, such as PCR, RNA-seq, and northern blot.

Combination Therapy

Some aspects of this disclosure are based on the recognition thatcertain disorders, e.g., certain proliferative diseases, can be moreeffectively treated by a combination therapy approach (e.g., byadministering an EZH2 inhibitor and an immune checkpoint inhibitor tothe subject) as compared to treatment with only a single therapeuticagent. For example, in some embodiments, the present disclosure providescombination therapy strategies, treatment modalities, methods,combinations, and compositions that are useful for improving theclinical outcome and/or the prognosis of a subject having aproliferative disease, e.g., a cancer, as compared to monotherapeuticapproaches. In some embodiments, the combination therapy approachesprovided herein result in a shorter time period being required toachieve a desired clinical outcome (e.g., partial or complete diseaseremission, inhibition of tumor growth, stable disease), as compared tomonotherapy. In some embodiments, the combination therapy approachesprovided herein result in a better clinical outcome as compared tomonotherapy (e.g., complete vs. partial remission, stable vs.progressive disease, lower recurrence risk).

In some embodiments, the present disclosure provides combination therapystrategies, treatment modalities, and methods, wherein a subject in needthereof is administered an EZH2 inhibitor and an immune checkpointinhibitor. In some embodiments, the EZH2 inhibitor is an EZH2 inhibitoras provided herein, e.g., a small molecule EZH2 inhibitor provided byany of Formulae (I)-(IVa), or by any other structure described herein,and the immune checkpoint inhibitor is a monoclonal antibody, a peptide,or a small molecule as described herein. In some embodiments, the EZH2inhibitor is tazemetostat, or a pharmaceutically acceptable saltthereof, and the immune checkpoint inhibitor is Ipilimumab, Ticilimumab,AGEN-1884, Nivolumab, Pembrolizumab, Atezolizumab, Durvalumab, Avelumab,BMS-936559, AMP-224, MEDI-0680, TSR-042, BGB-108, STI-1014, KY-1003,ALN-PDL, BGB-A317, KD-033, REGN-2810, PDR-001, SHR-1210, MGD-013,PF-06801591, CX-072, IMP-731, LAG-525, BMS-986016, GSK-2831781,Enoblituzumab, 1241-8H9, DS-5573, or a combination thereof.

In some embodiments, combination therapy strategies, treatmentmodalities, and methods for the treatment of proliferative diseases areprovided, wherein the EZH2 inhibitor is tazemetostat, or apharmaceutically acceptable salt thereof, and the immune checkpointinhibitor is Ipilimumab. For example, in some embodiments, a method isprovided that comprises administering to a subject in need thereof,e.g., a subject having or being diagnosed with a proliferative disease(e.g., a cancer), a therapeutically effective amount of tazemetostat, ora pharmaceutically acceptable salt thereof, and a therapeuticallyeffective amount of Ipilimumab.

In some embodiments, combination therapy strategies, treatmentmodalities, and methods for the treatment of proliferative diseases areprovided, wherein the EZH2 inhibitor is tazemetostat, or apharmaceutically acceptable salt thereof, and the immune checkpointinhibitor is Ticilimumab. For example, in some embodiments, a method isprovided that comprises administering to a subject in need thereof,e.g., a subject having or being diagnosed with a proliferative disease(e.g., a cancer), a therapeutically effective amount of tazemetostat, ora pharmaceutically acceptable salt thereof, and a therapeuticallyeffective amount of Ticilimumab.

In some embodiments, combination therapy strategies, treatmentmodalities, and methods for the treatment of proliferative diseases areprovided, wherein the EZH2 inhibitor is tazemetostat, or apharmaceutically acceptable salt thereof, and the immune checkpointinhibitor is AGEN-1884. For example, in some embodiments, a method isprovided that comprises administering to a subject in need thereof,e.g., a subject having or being diagnosed with a proliferative disease(e.g., a cancer), a therapeutically effective amount of tazemetostat, ora pharmaceutically acceptable salt thereof, and a therapeuticallyeffective amount of AGEN-1884.

In some embodiments, combination therapy strategies, treatmentmodalities, and methods for the treatment of proliferative diseases areprovided, wherein the EZH2 inhibitor is tazemetostat, or apharmaceutically acceptable salt thereof, and the immune checkpointinhibitor is Nivolumab. For example, in some embodiments, a method isprovided that comprises administering to a subject in need thereof,e.g., a subject having or being diagnosed with a proliferative disease(e.g., a cancer), a therapeutically effective amount of tazemetostat, ora pharmaceutically acceptable salt thereof, and a therapeuticallyeffective amount of Nivolumab.

In some embodiments, combination therapy strategies, treatmentmodalities, and methods for the treatment of proliferative diseases areprovided, wherein the EZH2 inhibitor is tazemetostat, or apharmaceutically acceptable salt thereof, and the immune checkpointinhibitor is Pembrolizumab. For example, in some embodiments, a methodis provided that comprises administering to a subject in need thereof,e.g., a subject having or being diagnosed with a proliferative disease(e.g., a cancer), a therapeutically effective amount of tazemetostat, ora pharmaceutically acceptable salt thereof, and a therapeuticallyeffective amount of Pembrolizumab.

In some embodiments, combination therapy strategies, treatmentmodalities, and methods for the treatment of proliferative diseases areprovided, wherein the EZH2 inhibitor is tazemetostat, or apharmaceutically acceptable salt thereof, and the immune checkpointinhibitor is Atezolizumab. For example, in some embodiments, a method isprovided that comprises administering to a subject in need thereof,e.g., a subject having or being diagnosed with a proliferative disease(e.g., a cancer), a therapeutically effective amount of tazemetostat, ora pharmaceutically acceptable salt thereof, and a therapeuticallyeffective amount of Atezolizumab.

In some embodiments, combination therapy strategies, treatmentmodalities, and methods for the treatment of proliferative diseases areprovided, wherein the EZH2 inhibitor is tazemetostat, or apharmaceutically acceptable salt thereof, and the immune checkpointinhibitor is Durvalumab. For example, in some embodiments, a method isprovided that comprises administering to a subject in need thereof,e.g., a subject having or being diagnosed with a proliferative disease(e.g., a cancer), a therapeutically effective amount of tazemetostat, ora pharmaceutically acceptable salt thereof, and a therapeuticallyeffective amount of Durvalumab.

In some embodiments, combination therapy strategies, treatmentmodalities, and methods for the treatment of proliferative diseases areprovided, wherein the EZH2 inhibitor is tazemetostat, or apharmaceutically acceptable salt thereof, and the immune checkpointinhibitor is Avelumab. For example, in some embodiments, a method isprovided that comprises administering to a subject in need thereof,e.g., a subject having or being diagnosed with a proliferative disease(e.g., a cancer), a therapeutically effective amount of tazemetostat, ora pharmaceutically acceptable salt thereof, and a therapeuticallyeffective amount of Avelumab.

In some embodiments, combination therapy strategies, treatmentmodalities, and methods for the treatment of proliferative diseases areprovided, wherein the EZH2 inhibitor is tazemetostat, or apharmaceutically acceptable salt thereof, and the immune checkpointinhibitor is BMS-936559. For example, in some embodiments, a method isprovided that comprises administering to a subject in need thereof,e.g., a subject having or being diagnosed with a proliferative disease(e.g., a cancer), a therapeutically effective amount of tazemetostat, ora pharmaceutically acceptable salt thereof, and a therapeuticallyeffective amount of BMS-936559.

In some embodiments, combination therapy strategies, treatmentmodalities, and methods for the treatment of proliferative diseases areprovided, wherein the EZH2 inhibitor is tazemetostat, or apharmaceutically acceptable salt thereof, and the immune checkpointinhibitor is AMP-224. For example, in some embodiments, a method isprovided that comprises administering to a subject in need thereof,e.g., a subject having or being diagnosed with a proliferative disease(e.g., a cancer), a therapeutically effective amount of tazemetostat, ora pharmaceutically acceptable salt thereof, and a therapeuticallyeffective amount of AMP-224.

In some embodiments, combination therapy strategies, treatmentmodalities, and methods for the treatment of proliferative diseases areprovided, wherein the EZH2 inhibitor is tazemetostat, or apharmaceutically acceptable salt thereof, and the immune checkpointinhibitor is MEDI-0680. For example, in some embodiments, a method isprovided that comprises administering to a subject in need thereof,e.g., a subject having or being diagnosed with a proliferative disease(e.g., a cancer), a therapeutically effective amount of tazemetostat, ora pharmaceutically acceptable salt thereof, and a therapeuticallyeffective amount of MEDI-0680.

In some embodiments, combination therapy strategies, treatmentmodalities, and methods for the treatment of proliferative diseases areprovided, wherein the EZH2 inhibitor is tazemetostat, or apharmaceutically acceptable salt thereof, and the immune checkpointinhibitor is Enoblituzumab. For example, in some embodiments, a methodis provided that comprises administering to a subject in need thereof,e.g., a subject having or being diagnosed with a proliferative disease(e.g., a cancer), a therapeutically effective amount of tazemetostat, ora pharmaceutically acceptable salt thereof, and a therapeuticallyeffective amount of Enoblituzumab.

In some embodiments, the method further includes monitoring themethylation status in a target cell or tissue in the subject, e.g., bymethods described herein or otherwise known to those in the art, and/ormonitoring the immune response status in the subject, e.g., by methodsdescribed herein or otherwise known in the art.

Pharmaceutical Formulations

In some embodiments, the EZH2 inhibitor and the immune checkpointinhibitor are provided in separate pharmaceutical formulations, andadministered to the subject independently, e.g., sequentially. In someembodiments, the EZH2 inhibitor is formulated for oral administrationand the immune response inhibitor is formulated for parenteraladministration.

The disclosure also provides pharmaceutical compositions andcombinations comprising a compound of Formulae (I)-(VIa) orpharmaceutically acceptable salts thereof, and one or more othertherapeutic agents disclosed herein, e.g., one or more immune checkpointinhibitors, mixed with pharmaceutically suitable carriers orexcipient(s) at doses to treat or prevent a disease or condition asdescribed herein. In one aspect, the disclosure also providespharmaceutical compositions comprising any compound of Table I orpharmaceutically acceptable salts thereof, and one or more therapeuticagents, mixed with pharmaceutically suitable carriers or excipient (s)at doses to treat or prevent a disease or condition as described herein.In another aspect, the disclosure also provides pharmaceuticalcompositions comprising Compound 44

or pharmaceutically acceptable salts thereof, and one or moretherapeutic agents, mixed with pharmaceutically suitable carriers orexcipient(s) at doses to treat or prevent a disease or condition asdescribed herein. The pharmaceutical compositions of the disclosure canalso be administered in combination with other therapeutic agents ortherapeutic modalities simultaneously, sequentially, or in alternation.

Mixtures or combinations of compositions of the disclosure can also beadministered to the patient as a simple mixture or in suitableformulated pharmaceutical compositions. For example, one aspect of theinvention relates to a pharmaceutical composition or combinationcomprising a therapeutically effective dose of an EZH2 inhibitor ofFormulae (I)-(VIa), or a pharmaceutically acceptable salt, hydrate,enantiomer or stereoisomer thereof; one or more other therapeuticagents, and a pharmaceutically acceptable diluent or carrier.

A “pharmaceutical composition” is a formulation containing the compoundsof the disclosure in a form suitable for administration to a subject. Acompound of Formulae (I)-(VIa) and one or more other therapeutic agentsdescribed herein each can be formulated individually or in multiplepharmaceutical compositions in any combinations of the activeingredients. Accordingly, one or more administration routes can beproperly elected based on the dosage form of each pharmaceuticalcomposition. Alternatively, a compound of Formulae (I)-(VIa) and one ormore other therapeutic agents described herein can be formulated as onepharmaceutical composition.

In some embodiments, the pharmaceutical composition is in bulk or inunit dosage form. The unit dosage form is any of a variety of forms,including, for example, a capsule, an IV bag, a tablet, a single pump onan aerosol inhaler or a vial. The quantity of active ingredient (e.g., aformulation of the disclosed compound or salt, hydrate, solvate orisomer thereof) in a unit dose of composition is an effective amount andis varied according to the particular treatment involved. One skilled inthe art will appreciate that it is sometimes necessary to make routinevariations to the dosage depending on the age and condition of thepatient. The dosage will also depend on the route of administration. Avariety of routes are contemplated, including oral, pulmonary, rectal,parenteral, transdermal, subcutaneous, intravenous, intramuscular,intraperitoneal, inhalational, buccal, sublingual, intrapleural,intrathecal, intranasal, and the like. Dosage forms for the topical ortransdermal administration of a compound of this disclosure includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches and inhalants. In some embodiments, the active compound is mixedunder sterile conditions with a pharmaceutically acceptable carrier, andwith any preservatives, buffers, or propellants that are required.

As used herein, the phrase “pharmaceutically acceptable” refers to thosecompounds, anions, cations, materials, compositions, carriers, and/ordosage forms which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of human beings and animalswithout excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio.

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic and neither biologically nor otherwise undesirable, andincludes excipient that is acceptable for veterinary use as well ashuman pharmaceutical use. A “pharmaceutically acceptable excipient” asused in the specification and claims includes both one and more than onesuch excipient.

A pharmaceutical composition of the disclosure is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical), andtransmucosal administration. Solutions or suspensions used forparenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates, and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

A composition of the disclosure, e.g., a formulation comprising an EZH2inhibitor and/or an immune checkpoint inhibitor can be administered to asubject in many of the well-known methods currently used forchemotherapeutic treatment. For example, for treatment of cancers, aformulation comprising an EZH2 inhibitor and/or an immune checkpointinhibitor be injected directly into tumors, injected into the bloodstream or body cavities or taken orally or applied through the skin withpatches. The dose chosen for the EZH2 inhibitor and for the immunecheckpoint inhibitor should be sufficient to constitute effectivetreatment but not so high as to cause unacceptable side effects. Thestate of the disease condition (e.g., cancer, precancer, and the like)and the health of the patient should preferably be closely monitoredduring and for a reasonable period after treatment.

The term “therapeutically effective amount”, as used herein, refers toan amount of a pharmaceutical agent to treat, ameliorate, or prevent anidentified disease or condition, or to exhibit a detectable therapeuticor inhibitory effect. Exemplary, non-limiting effective amounts andeffective dosage ranges of EZH2 inhibitors and immune responseinhibitors are provided herein. The effect can be detected by any assaymethod known in the art. The precise effective amount for a subject willdepend upon the subject's body weight, size, and health; the nature andextent of the condition; and the therapeutic or combination oftherapeutics selected for administration. Therapeutically effectiveamounts for a given situation can be determined by routineexperimentation that is within the skill and judgment of the clinician.In a preferred aspect, the disease or condition to be treated is cancer.In another aspect, the disease or condition to be treated is a cellproliferative disorder.

In certain embodiments the therapeutically effective amount of eachpharmaceutical agent used in combination will be lower when used incombination in comparison to monotherapy with each agent alone. Suchlower therapeutically effective amount could afford for lower toxicityof the therapeutic regimen.

For many of the compounds described herein, e.g., various EZH2inhibitors and various immune checkpoint inhibitors, a therapeuticallyeffective amount or an effective dosage range has been reported. In someembodiments, an effective amount can be estimated initially either incell culture assays, e.g., of neoplastic cells, or in animal models,usually rats, mice, rabbits, dogs, or pigs. The animal model may also beused to determine the appropriate concentration range and route ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans. Therapeutic/prophylacticefficacy and toxicity may be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., ED₅₀ (thedose therapeutically effective in 50% of the population) and LD₅₀ (thedose lethal to 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index, and it can be expressed asthe ratio, LD₅₀/ED₅₀. Pharmaceutical compositions that exhibit largetherapeutic indices are preferred. The dosage may vary within this rangedepending upon the dosage form employed, sensitivity of the patient, andthe route of administration.

Dosage and administration are adjusted to provide sufficient levels ofthe active agent(s) or to maintain the desired effect. Factors which maybe taken into account include the severity of the disease state, generalhealth of the subject, age, weight, and gender of the subject, diet,time and frequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

The pharmaceutical compositions containing active compounds of thedisclosure may be manufactured in a manner that is generally known,e.g., by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping, orlyophilizing processes. Pharmaceutical compositions may be formulated ina conventional manner using one or more pharmaceutically acceptablecarriers comprising excipients and/or auxiliaries that facilitateprocessing of the active compounds into preparations that can be usedpharmaceutically. Of course, the appropriate formulation is dependentupon the route of administration chosen.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol and sorbitol, and sodium chloridein the composition. Prolonged absorption of the injectable compositionscan be brought about by including in the composition an agent whichdelays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblepharmaceutically acceptable carrier. They can be enclosed in gelatincapsules or compressed into tablets. For the purpose of oral therapeuticadministration, the active compound can be incorporated with excipientsand used in the form of tablets, troches, or capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash,wherein the compound in the fluid carrier is applied orally and swishedand expectorated or swallowed. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition. The tablets, pills, capsules, troches and the like cancontain any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a disintegrating agentsuch as alginic acid, Primogel, or corn starch; a lubricant such asmagnesium stearate or Sterotes; a glidant such as colloidal silicondioxide; a sweetening agent such as sucrose or saccharin; or a flavoringagent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser, whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The active compounds can be prepared with pharmaceutically acceptablecarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the disclosure are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved.

In some embodiments of therapeutic applications, the dosages of the EZH2inhibitors and/or the immune checkpoint inhibitors described herein,e.g., compositions comprising a compound of Formulae (I)-(VIa),tazemetostat, and/or an immune checkpoint inhibitor, or thepharmaceutical compositions used in accordance with the disclosure, varydepending on the specific agent(s) used, the age, weight, and clinicalcondition of the recipient patient, and the experience and judgment ofthe clinician or practitioner administering the therapy, among otherfactors affecting the selected dosage. Generally, the dose of the activeingredient(s) should be sufficient to result in slowing, and preferablyregressing, the growth of the tumors and also preferably causingcomplete regression of the cancer. In some embodiments, dosages canrange from about 0.01 mg/kg per day to about 5000 mg/kg per day. Inpreferred aspects, dosages can range from about 1 mg/kg per day to about1000 mg/kg per day. In an aspect, the dose will be in the range of about0.1 mg/day to about 50 g/day; about 0.1 mg/day to about 25 g/day; about0.1 mg/day to about 10 g/day; about 0.1 mg to about 3 g/day; or about0.1 mg to about 1 g/day, in single, divided, or continuous doses (whichdose may be adjusted for the patient's weight in kg, body surface areain m², and age in years). Additional suitable dosages are providedelsewhere herein. For example, regression of a tumor in a patient may bemeasured with reference to the diameter of a tumor. Decrease in thediameter of a tumor indicates regression. Regression is also indicatedby failure of tumors to reoccur after treatment has stopped. As usedherein, the term “dosage effective manner” refers to amount of an activecompound to produce the desired biological effect in a subject or cell.

As used herein, “pharmaceutically acceptable salts” refer to derivativesof the compounds of the disclosure, e.g., of the small molecule EZH2inhibitors described herein, wherein the parent compound is modified bymaking acid or base salts thereof. Examples of pharmaceuticallyacceptable salts, e.g., of the EZH2 inhibitors provided herein, include,but are not limited to, mineral or organic acid salts of basic residuessuch as amines, alkali or organic salts of acidic residues such ascarboxylic acids, and the like. The pharmaceutically acceptable saltsinclude the conventional non-toxic salts or the quaternary ammoniumsalts of the parent compound formed, for example, from non-toxicinorganic or organic acids. For example, such conventional non-toxicsalts include, but are not limited to, those derived from inorganic andorganic acids selected from 2-acetoxybenzoic, 2-hydroxyethane sulfonic,acetic, ascorbic, benzene sulfonic, benzoic, bicarbonic, carbonic,citric, edetic, ethane disulfonic, 1,2-ethane sulfonic, fumaric,glucoheptonic, gluconic, glutamic, glycolic, glycollyarsanilic,hexylresorcinic, hydrabamic, hydrobromic, hydrochloric, hydroiodic,hydroxymaleic, hydroxynaphthoic, isethionic, lactic, lactobionic, laurylsulfonic, maleic, malic, mandelic, methane sulfonic, napsylic, nitric,oxalic, pamoic, pantothenic, phenylacetic, phosphoric, polygalacturonic,propionic, salicyclic, stearic, subacetic, succinic, sulfamic,sulfanilic, sulfuric, tannic, tartaric, toluene sulfonic, and thecommonly occurring amine acids, e.g., glycine, alanine, phenylalanine,arginine, etc.

Other examples of pharmaceutically acceptable salts include hexanoicacid, cyclopentane propionic acid, pyruvic acid, malonic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, 4-chlorobenzenesulfonicacid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid,camphorsulfonic acid, 4-methylbicyclo-[2.2.2]-oct-2-ene-1-carboxylicacid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylaceticacid, muconic acid, and the like. The disclosure also encompasses saltsformed when an acidic proton present in the parent compound either isreplaced by a metal ion, e.g., an alkali metal ion, an alkaline earthion, or an aluminum ion; or coordinates with an organic base such asethanolamine, diethanolamine, triethanolamine, tromethamine,N-methylglucamine, and the like.

It should be understood that all references to pharmaceuticallyacceptable salts include solvent addition forms (solvates), of the samesalt.

The composition of the disclosure may also be prepared as esters, forexample, pharmaceutically acceptable esters. For example, a carboxylicacid function group in a compound can be converted to its correspondingester, e.g., a methyl, ethyl or other ester. Also, an alcohol group in acompound can be converted to its corresponding ester, e.g., acetate,propionate or other ester.

The composition, or pharmaceutically acceptable salts or solvatesthereof, are administered orally, nasally, transdermally, pulmonary,inhalationally, buccally, sublingually, intraperintoneally,subcutaneously, intramuscularly, intravenously, rectally,intraperitoneally, intrathecally and parenterally. In some embodiments,the compound is administered orally. One skilled in the art willrecognize the advantages of certain routes of administration.

The dosage regimen utilizing the compounds is selected in accordancewith a variety of factors including type, species, age, weight, sex andmedical condition of the patient; the severity of the condition to betreated; the route of administration; the renal and hepatic function ofthe patient; and the particular compound or salt thereof employed. Anordinarily skilled physician or veterinarian can readily determine andprescribe the effective amount of the drug required to prevent, counter,or arrest the progress of the condition.

Techniques for formulation and administration of the disclosed compoundsof the disclosure can be found in Remington: the Science and Practice ofPharmacy, 19^(th) edition, Mack Publishing Co., Easton, Pa. (1995). Insome embodiments, the compounds described herein, and thepharmaceutically acceptable salts thereof, are used in pharmaceuticalpreparations in combination with a pharmaceutically acceptable carrieror diluent. Suitable pharmaceutically acceptable carriers include inertsolid fillers or diluents and sterile aqueous or organic solutions. Thecompounds will be present in such pharmaceutical compositions in amountssufficient to provide the desired dosage amount in the range describedherein.

All percentages and ratios used herein, unless otherwise indicated, areby weight. Other features and advantages of the disclosure are apparentfrom the different examples. The provided examples illustrate differentcomponents and methodology useful in practicing the disclosure. Theexamples do not limit the claimed disclosure. Based on the presentdisclosure the skilled artisan can identify and employ other componentsand methodology useful for practicing the disclosure.

In some embodiments, a “subject in need thereof” is a subject having adisorder in which EZH2-mediated protein methylation plays a part, or asubject having an increased risk of developing such disorder relative tothe population at large. In some embodiments, the subject has a disorderin which immune system evasion also plays a role, e.g., immune systemevasion of cancer cells via immune checkpoint signaling. In someembodiments, a subject in need thereof has a proliferative disease,e.g., a cancer. A “subject” includes a mammal. The mammal can be e.g.,any mammal, e.g., a human, primate, bird, mouse, rat, fowl, dog, cat,cow, horse, goat, camel, sheep or a pig. Preferably, the mammal is ahuman.

In some embodiments, the subject is a human subject who has beendiagnosed with, has symptoms of, or is at risk of developing a cancer ora precancerous condition. In some embodiments, the subject expresses amutant EZH2 protein. For example, a mutant EZH2 comprising one or moremutations, wherein the mutation is a substitution, a point mutation, anonsense mutation, a missense mutation, a deletion, or an insertion orany other EZH2 mutation described herein. In some embodiments, thesubject expresses a wild type EZH2 protein.

A subject in need thereof may have refractory or resistant cancer.“Refractory or resistant cancer” means cancer that does not respond totreatment, e.g., to treatment with a monotherapy, e.g., a monotherapywith an immune checkpoint inhibitor alone or with an EZH2 inhibitoralone. In some embodiments, the cancer may be refractory or resistant tothe standard of care treatment for that particular type of cancer. Thecancer may be resistant at the beginning of treatment or it may becomeresistant during treatment. In some embodiments, the subject in needthereof has cancer recurrence following remission on most recenttherapy. In some embodiments, the subject in need thereof received andfailed all known effective therapies for cancer treatment. In someembodiments, the subject in need thereof received at least one priortherapy. In certain embodiments the prior therapy is monotherapy. Incertain embodiments the prior therapy is combination therapy.

In some embodiments, a subject in need thereof may have a secondarycancer as a result of a previous therapy. “Secondary cancer” meanscancer that arises due to or as a result from previous carcinogenictherapies, such as chemotherapy.

The subject may also exhibit resistance to EZH2 histonemethyltransferase inhibitors or any other therapeutic agent.

As used herein, the term “responsiveness” is interchangeable with terms“responsive”, “sensitive”, and “sensitivity”, and it is meant that asubject is showing therapeutic responses when administered a compositionof the disclosure, e.g., tumor cells or tumor tissues of the subjectundergo apoptosis and/or necrosis, and/or display reduced growing,dividing, or proliferation. This term is also meant that a subject willor has a higher probability, relative to the population at large, ofshowing therapeutic responses when administered a composition of thedisclosure, e.g., tumor cells or tumor tissues of the subject undergoapoptosis and/or necrosis, and/or display reduced growing, dividing, orproliferation.

The term “sample” refers to any biological sample derived from thesubject, includes but is not limited to, cells, tissues samples, bodyfluids (including, but not limited to, mucus, blood, plasma, serum,urine, saliva, and semen), tumor cells, and tumor tissues. Preferably,the sample is selected from bone marrow, peripheral blood cells, blood,plasma and serum. Samples can be provided by the subject under treatmentor testing. Alternatively samples can be obtained by the physicianaccording to routine practice in the art.

As used herein, a “normal cell” is a cell that cannot be classified aspart of a “cell proliferative disorder”. A normal cell lacks unregulatedor abnormal growth, or both, that can lead to the development of anunwanted condition or disease. Preferably, a normal cell possessesnormally functioning cell cycle checkpoint control mechanisms.

As used herein, “contacting a cell” refers to a condition in which acompound or other composition of matter is in direct contact with acell, or is close enough to induce a desired biological effect in acell.

As used herein, “treating” or “treat” describes the management and careof a patient for the purpose of combating a disease, condition, ordisorder and includes the administration of an EZH2 inhibitor and/or animmune checkpoint inhibitor, to alleviate the symptoms or complicationsof a disease, condition or disorder, or to eliminate the disease,condition or disorder.

Cancer

A “cancer cell” or “cancerous cell” is a cell manifesting a cellproliferative disorder that is a cancer. Any reproducible means ofmeasurement may be used to identify cancer cells or precancerous cells.Cancer cells or precancerous cells can be identified by histologicaltyping or grading of a tissue sample (e.g., a biopsy sample). Cancercells or precancerous cells can be identified through the use ofappropriate molecular markers.

Exemplary cancers suitable for the strategies, treatment modalities,methods, combinations, and compositions provided herein include, but arenot limited to, adrenocortical carcinoma, AIDS-related cancers,AIDS-related lymphoma, anal cancer, anorectal cancer, cancer of the analcanal, appendix cancer, childhood cerebellar astrocytoma, childhoodcerebral astrocytoma, basal cell carcinoma, skin cancer (non-melanoma),biliary cancer, extrahepatic bile duct cancer, intrahepatic bile ductcancer, bladder cancer, urinary bladder cancer, bone and joint cancer,osteosarcoma and malignant fibrous histiocytoma, brain cancer, braintumor, brain stem glioma, cerebellar astrocytoma, cerebralastrocytoma/malignant glioma, ependymoma, medulloblastoma,supratentorial primitive neuroectodermal tumors, visual pathway andhypothalamic glioma, breast cancer, bronchial adenomas/carcinoids,carcinoid tumor, gastrointestinal, nervous system cancer, nervous systemlymphoma, central nervous system cancer, central nervous systemlymphoma, cervical cancer, childhood cancers, chronic lymphocyticleukemia, chronic myelogenous leukemia, chronic myeloproliferativedisorders, colon cancer, colorectal cancer, cutaneous T-cell lymphoma,lymphoid neoplasm, mycosis fungoides, Sezary Syndrome, endometrialcancer, esophageal cancer, extracranial germ cell tumor, extragonadalgerm cell tumor, extrahepatic bile duct cancer, eye cancer, intraocularmelanoma, retinoblastoma, gallbladder cancer, gastric (stomach) cancer,gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST),germ cell tumor, ovarian germ cell tumor, gestational trophoblastictumor glioma, head and neck cancer, hepatocellular (liver) cancer,Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, ocularcancer, islet cell tumors (endocrine pancreas), Kaposi Sarcoma, kidneycancer, renal cancer, kidney cancer, laryngeal cancer, acutelymphoblastic leukemia, acute myeloid leukemia, chronic lymphocyticleukemia, chronic myelogenous leukemia, hairy cell leukemia, lip andoral cavity cancer, liver cancer, lung cancer, non-small cell lungcancer, small cell lung cancer, AIDS-related lymphoma, non-Hodgkinlymphoma, primary central nervous system lymphoma, Waldenstroemmacroglobulinemia, melanoma, intraocular (eye) melanoma, merkel cellcarcinoma, mesothelioma malignant, mesothelioma, metastatic squamousneck cancer, mouth cancer, cancer of the tongue, multiple endocrineneoplasia syndrome, mycosis fungoides, myelodysplastic syndromes,myelodysplastic/myeloproliferative diseases, chronic myelogenousleukemia, acute myeloid leukemia, multiple myeloma, chronicmyeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, oralcancer, oral cavity cancer, oropharyngeal cancer, ovarian cancer,ovarian epithelial cancer, ovarian low malignant potential tumor,ovarian clear cell adenocarcinoma, ovarian endometrioid adenocarcinoma,ovarian serous adenocarcinoma, pancreatic ductal adenocarcinoma,pancreatic endocrine tumor, pancreatic cancer, islet cell pancreaticcancer, paranasal sinus and nasal cavity cancer, parathyroid cancer,penile cancer, pharyngeal cancer, pheochromocytoma, pineoblastoma andsupratentorial primitive neuroectodermal tumors, pituitary tumor, plasmacell neoplasm/multiple myeloma, pleuropulmonary blastoma, prostatecancer, rectal cancer, renal pelvis and ureter, transitional cellcancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, ewingfamily of sarcoma tumors, Kaposi Sarcoma, soft tissue sarcoma, synovialsarcoma, uterine cancer, uterine sarcoma, skin cancer (non-melanoma),skin cancer (melanoma), merkel cell skin carcinoma, small intestinecancer, soft tissue sarcoma, squamous cell carcinoma, stomach (gastric)cancer, supratentorial primitive neuroectodermal tumors, testicularcancer, throat cancer, thymoma, thymoma and thymic carcinoma, thyroidcancer, transitional cell cancer of the renal pelvis and ureter andother urinary organs, gestational trophoblastic tumor, urethral cancer,endometrial uterine cancer, uterine sarcoma, uterine corpus cancer,vaginal cancer, vulvar cancer, Wilm's Tumor, malignant rhabdoid tumor,astrocytoma, atypical teratoid rhabdoid tumor, choroid plexus carcinoma,choroid plexus papilloma, ependymoma, glioblastoma, meningioma,neuroglial tumor, oligoastrocytoma, oligodendroglioma, carcinosarcoma,chordoma, extrarenal rhabdoid tumor, schwannoma, skin squamous cellcarcinoma, chondrosarcoma, clear cell sarcoma of soft tissue, ewingsarcoma, epithelioid sarcoma, renal medullary carcinoma, diffuse largeB-cell lymphoma, follicular lymphoma and not otherwise specified (NOS)sarcoma.

A “cell proliferative disorder of the hematologic system” is a cellproliferative disorder involving cells of the hematologic system. A cellproliferative disorder of the hematologic system suitable for thestrategies, treatment modalities, methods, combinations, andcompositions provided herein can include lymphoma, leukemia, myeloidneoplasms, mast cell neoplasms, myelodysplasia, benign monoclonalgammopathy, lymphomatoid granulomatosis, lymphomatoid papulosis,polycythemia vera, chronic myelocytic leukemia, agnogenic myeloidmetaplasia, and essential thrombocythemia. A cell proliferative disorderof the hematologic system can include hyperplasia, dysplasia, andmetaplasia of cells of the hematologic system. In some embodiments, thestrategies, treatment modalities, methods, combinations, andcompositions provided herein are used to treat a cancer selected fromthe group consisting of a hematologic cancer of the disclosure or ahematologic cell proliferative disorder of the disclosure. A hematologiccancer of the disclosure can include multiple myeloma, lymphoma(including Hodgkin's lymphoma, non-Hodgkin's lymphoma, childhoodlymphomas, and lymphomas of lymphocytic and cutaneous origin), leukemia(including childhood leukemia, hairy-cell leukemia, acute lymphocyticleukemia, acute myelocytic leukemia, chronic lymphocytic leukemia,chronic myelocytic leukemia, chronic myelogenous leukemia, and mast cellleukemia), myeloid neoplasms and mast cell neoplasms.

A “cell proliferative disorder of the lung” suitable for the strategies,treatment modalities, methods, combinations, and compositions providedherein includes a cell proliferative disorder involving cells of thelung. Cell proliferative disorders of the lung can include all forms ofcell proliferative disorders affecting lung cells. Cell proliferativedisorders of the lung can include lung cancer, a precancer orprecancerous condition of the lung, benign growths or lesions of thelung, and malignant growths or lesions of the lung, and metastaticlesions in tissue and organs in the body other than the lung. Cellproliferative disorders of the lung can include hyperplasia, metaplasia,and dysplasia of the lung. In some embodiments, the strategies,treatment modalities, methods, combinations, and compositions providedherein are used to treat lung cancer or cell proliferative disorders ofthe lung. Lung cancer can include all forms of cancer of the lung. Lungcancer can include malignant lung neoplasms, carcinoma in situ, typicalcarcinoid tumors, and atypical carcinoid tumors. Lung cancer can includesmall cell lung cancer (“SCLC”), non-small cell lung cancer (“NSCLC”),squamous cell carcinoma, adenocarcinoma, small cell carcinoma, largecell carcinoma, adenosquamous cell carcinoma, and mesothelioma. Lungcancer can include “scar carcinoma,” bronchioalveolar carcinoma, giantcell carcinoma, spindle cell carcinoma, and large cell neuroendocrinecarcinoma. Lung cancer can include lung neoplasms having histologic andultrastructural heterogeneity (e.g., mixed cell types).

Cell proliferative disorders of the lung can also includeasbestos-induced hyperplasia, squamous metaplasia, and benign reactivemesothelial metaplasia. Cell proliferative disorders of the lung caninclude replacement of columnar epithelium with stratified squamousepithelium, and mucosal dysplasia. Individuals exposed to inhaledinjurious environmental agents such as cigarette smoke and asbestos maybe at increased risk for developing cell proliferative disorders of thelung. Prior lung diseases that may predispose individuals to developmentof cell proliferative disorders of the lung can include chronicinterstitial lung disease, necrotizing pulmonary disease, scleroderma,rheumatoid disease, sarcoidosis, interstitial pneumonitis, tuberculosis,repeated pneumonias, idiopathic pulmonary fibrosis, granulomata,asbestosis, fibrosing alveolitis, and Hodgkin's disease.

A “cell proliferative disorder of the colon” is a cell proliferativedisorder involving cells of the colon. In some embodiments, the cellproliferative disorder of the colon is colon cancer. In someembodiments, the strategies, treatment modalities, methods,combinations, and compositions provided herein are used to treat coloncancer or cell proliferative disorders of the colon. Colon cancer caninclude all forms of cancer of the colon. Colon cancer can includesporadic and hereditary colon cancers. Colon cancer can includemalignant colon neoplasms, carcinoma in situ, typical carcinoid tumors,and atypical carcinoid tumors. Colon cancer can include adenocarcinoma,squamous cell carcinoma, and adenosquamous cell carcinoma. Colon cancercan be associated with a hereditary syndrome selected from the groupconsisting of hereditary nonpolyposis colorectal cancer, familialadenomatous polyposis, Gardner's syndrome, Peutz-Jeghers syndrome,Turcot's syndrome and juvenile polyposis. Colon cancer can be caused bya hereditary syndrome selected from the group consisting of hereditarynonpolyposis colorectal cancer, familial adenomatous polyposis,Gardner's syndrome, Peutz-Jeghers syndrome, Turcot's syndrome andjuvenile polyposis.

Cell proliferative disorders of the colon can include all forms of cellproliferative disorders affecting colon cells. Cell proliferativedisorders of the colon can include colon cancer, precancerous conditionsof the colon, adenomatous polyps of the colon and metachronous lesionsof the colon. A cell proliferative disorder of the colon can includeadenoma. Cell proliferative disorders of the colon can be characterizedby hyperplasia, metaplasia, and dysplasia of the colon. Prior colondiseases that may predispose individuals to development of cellproliferative disorders of the colon can include prior colon cancer.Current disease that may predispose individuals to development of cellproliferative disorders of the colon can include Crohn's disease andulcerative colitis. A cell proliferative disorder of the colon can beassociated with a mutation in a gene selected from the group consistingof p53, ras, FAP and DCC. An individual can have an elevated risk ofdeveloping a cell proliferative disorder of the colon due to thepresence of a mutation in a gene selected from the group consisting ofp53, ras, FAP and DCC.

A “cell proliferative disorder of the pancreas” is a cell proliferativedisorder involving cells of the pancreas. Cell proliferative disordersof the pancreas can include all forms of cell proliferative disordersaffecting pancreatic cells. Cell proliferative disorders of the pancreascan include pancreas cancer, a precancer or precancerous condition ofthe pancreas, hyperplasia of the pancreas, and dysplasia of thepancreas, benign growths or lesions of the pancreas, and malignantgrowths or lesions of the pancreas, and metastatic lesions in tissue andorgans in the body other than the pancreas. Pancreatic cancer includesall forms of cancer of the pancreas. Pancreatic cancer can includeductal adenocarcinoma, adenosquamous carcinoma, pleomorphic giant cellcarcinoma, mucinous adenocarcinoma, osteoclast-like giant cellcarcinoma, mucinous cystadenocarcinoma, acinar carcinoma, unclassifiedlarge cell carcinoma, small cell carcinoma, pancreatoblastoma, papillaryneoplasm, mucinous cystadenoma, papillary cystic neoplasm, and serouscystadenoma. Pancreatic cancer can also include pancreatic neoplasmshaving histologic and ultrastructural heterogeneity (e.g., mixed celltypes).

A “cell proliferative disorder of the prostate” is a cell proliferativedisorder involving cells of the prostate. Cell proliferative disordersof the prostate can include all forms of cell proliferative disordersaffecting prostate cells. Cell proliferative disorders of the prostatecan include prostate cancer, a precancer or precancerous condition ofthe prostate, benign growths or lesions of the prostate, malignantgrowths or lesions of the prostate and metastatic lesions in tissue andorgans in the body other than the prostate. Cell proliferative disordersof the prostate can include hyperplasia, metaplasia, and dysplasia ofthe prostate.

A “cell proliferative disorder of the skin” is a cell proliferativedisorder involving cells of the skin. Cell proliferative disorders ofthe skin can include all forms of cell proliferative disorders affectingskin cells. Cell proliferative disorders of the skin can include aprecancer or precancerous condition of the skin, benign growths orlesions of the skin, melanoma, malignant melanoma and other malignantgrowths or lesions of the skin, and metastatic lesions in tissue andorgans in the body other than the skin. Cell proliferative disorders ofthe skin can include hyperplasia, metaplasia, and dysplasia of the skin.

A “cell proliferative disorder of the ovary” is a cell proliferativedisorder involving cells of the ovary. Cell proliferative disorders ofthe ovary can include all forms of cell proliferative disordersaffecting cells of the ovary. Cell proliferative disorders of the ovarycan include a precancer or precancerous condition of the ovary, benigngrowths or lesions of the ovary, ovarian cancer, malignant growths orlesions of the ovary, and metastatic lesions in tissue and organs in thebody other than the ovary. Cell proliferative disorders of the skin caninclude hyperplasia, metaplasia, and dysplasia of cells of the ovary.

A “cell proliferative disorder of the breast” is a cell proliferativedisorder involving cells of the breast. Cell proliferative disorders ofthe breast can include all forms of cell proliferative disordersaffecting breast cells. Cell proliferative disorders of the breast caninclude breast cancer, a precancer or precancerous condition of thebreast, benign growths or lesions of the breast, and malignant growthsor lesions of the breast, and metastatic lesions in tissue and organs inthe body other than the breast. Cell proliferative disorders of thebreast can include hyperplasia, metaplasia, and dysplasia of the breast.

A cell proliferative disorder of the breast can be a precancerouscondition of the breast. In some embodiments, the strategies, treatmentmodalities, methods, combinations, and compositions provided herein areused to treat a precancerous condition of the breast. A precancerouscondition of the breast can include atypical hyperplasia of the breast,ductal carcinoma in situ (DCIS), intraductal carcinoma, lobularcarcinoma in situ (LCIS), lobular neoplasia, and stage 0 or grade 0growth or lesion of the breast (e.g., stage 0 or grade 0 breast cancer,or carcinoma in situ). A precancerous condition of the breast can bestaged according to the TNM classification scheme as accepted by theAmerican Joint Committee on Cancer (AJCC), where the primary tumor (T)has been assigned a stage of T0 or Tis; and where the regional lymphnodes (N) have been assigned a stage of N0; and where distant metastasis(M) has been assigned a stage of M0.

The cell proliferative disorder of the breast can be breast cancer.Preferably, compositions of the disclosure may be used to treat breastcancer. Breast cancer includes all forms of cancer of the breast. Breastcancer can include primary epithelial breast cancers. Breast cancer caninclude cancers in which the breast is involved by other tumors such aslymphoma, sarcoma or melanoma. Breast cancer can include carcinoma ofthe breast, ductal carcinoma of the breast, lobular carcinoma of thebreast, undifferentiated carcinoma of the breast, cystosarcoma phyllodesof the breast, angiosarcoma of the breast, and primary lymphoma of thebreast. Breast cancer can include Stage I, II, IIIA, IIIB, IIIC and IVbreast cancer. Ductal carcinoma of the breast can include invasivecarcinoma, invasive carcinoma in situ with predominant intraductalcomponent, inflammatory breast cancer, and a ductal carcinoma of thebreast with a histologic type selected from the group consisting ofcomedo, mucinous (colloid), medullary, medullary with lymphocyticinfiltrate, papillary, scirrhous, and tubular. Lobular carcinoma of thebreast can include invasive lobular carcinoma with predominant in situcomponent, invasive lobular carcinoma, and infiltrating lobularcarcinoma. Breast cancer can include Paget's disease, Paget's diseasewith intraductal carcinoma, and Paget's disease with invasive ductalcarcinoma. Breast cancer can include breast neoplasms having histologicand ultrastructural heterogeneity (e.g., mixed cell types).

In some embodiments, the strategies, treatment modalities, methods,combinations, and compositions provided herein are used to treat breastcancer. A breast cancer that is to be treated can include familialbreast cancer. A breast cancer that is to be treated can includesporadic breast cancer. A breast cancer that is to be treated can arisein a male subject. A breast cancer that is to be treated can arise in afemale subject. A breast cancer that is to be treated can arise in apremenopausal female subject or a postmenopausal female subject. Abreast cancer that is to be treated can arise in a subject equal to orolder than 30 years old, or a subject younger than 30 years old. Abreast cancer that is to be treated has arisen in a subject equal to orolder than 50 years old, or a subject younger than 50 years old. Abreast cancer that is to be treated can arise in a subject equal to orolder than 70 years old, or a subject younger than 70 years old.

A breast cancer that is to be treated can be typed to identify afamilial or spontaneous mutation in BRCA1, BRCA2, or p53. A breastcancer that is to be treated can be typed as having a HER2/neu geneamplification, as overexpressing HER2/neu, or as having a low,intermediate or high level of HER2/neu expression. A breast cancer thatis to be treated can be typed for a marker selected from the groupconsisting of estrogen receptor (ER), progesterone receptor (PR), humanepidermal growth factor receptor-2, Ki-67, CA15-3, CA 27-29, and c-Met.A breast cancer that is to be treated can be typed as ER-unknown,ER-rich or ER-poor. A breast cancer that is to be treated can be typedas ER-negative or ER-positive. ER-typing of a breast cancer may beperformed by any reproducible means. ER-typing of a breast cancer may beperformed as set forth in Onkologie 27: 175-179 (2004). A breast cancerthat is to be treated can be typed as PR-unknown, PR-rich, or PR-poor. Abreast cancer that is to be treated can be typed as PR-negative orPR-positive. A breast cancer that is to be treated can be typed asreceptor positive or receptor negative. A breast cancer that is to betreated can be typed as being associated with elevated blood levels ofCA 15-3, or CA 27-29, or both.

A breast cancer that is to be treated can include a localized tumor ofthe breast. A breast cancer that is to be treated can include a tumor ofthe breast that is associated with a negative sentinel lymph node (SLN)biopsy. A breast cancer that is to be treated can include a tumor of thebreast that is associated with a positive sentinel lymph node (SLN)biopsy. A breast cancer that is to be treated can include a tumor of thebreast that is associated with one or more positive axillary lymphnodes, where the axillary lymph nodes have been staged by any applicablemethod. A breast cancer that is to be treated can include a tumor of thebreast that has been typed as having nodal negative status (e.g.,node-negative) or nodal positive status (e.g., node-positive). A breastcancer that is to be treated can include a tumor of the breast that hasmetastasized to other locations in the body. A breast cancer that is tobe treated can be classified as having metastasized to a locationselected from the group consisting of bone, lung, liver, or brain. Abreast cancer that is to be treated can be classified according to acharacteristic selected from the group consisting of metastatic,localized, regional, local-regional, locally advanced, distant,multicentric, bilateral, ipsilateral, contralateral, newly diagnosed,recurrent, and inoperable.

In some embodiments, the strategies, treatment modalities, methods,combinations, and compositions provided herein are used to treat orprevent a cell proliferative disorder of the breast, or to treat orprevent breast cancer, in a subject having an increased risk ofdeveloping breast cancer relative to the population at large. A subjectwith an increased risk of developing breast cancer relative to thepopulation at large is a female subject with a family history orpersonal history of breast cancer. A subject with an increased risk ofdeveloping breast cancer relative to the population at large is a femalesubject having a germ-line or spontaneous mutation in BRCA1 or BRCA2, orboth. A subject with an increased risk of developing breast cancerrelative to the population at large is a female subject with a familyhistory of breast cancer and a germ-line or spontaneous mutation inBRCA1 or BRCA2, or both. A subject with an increased risk of developingbreast cancer relative to the population at large is a female who isgreater than 30 years old, greater than 40 years old, greater than 50years old, greater than 60 years old, greater than 70 years old, greaterthan 80 years old, or greater than 90 years old. A subject with anincreased risk of developing breast cancer relative to the population atlarge is a subject with atypical hyperplasia of the breast, ductalcarcinoma in situ (DCIS), intraductal carcinoma, lobular carcinoma insitu (LCIS), lobular neoplasia, or a stage 0 growth or lesion of thebreast (e.g., stage 0 or grade 0 breast cancer, or carcinoma in situ).

A breast cancer that is to be treated can histologically gradedaccording to the Scarff-Bloom-Richardson system, wherein a breast tumorhas been assigned a mitosis count score of 1, 2, or 3; a nuclearpleomorphism score of 1, 2, or 3; a tubule formation score of 1, 2, or3; and a total Scarff-Bloom-Richardson score of between 3 and 9. Abreast cancer that is to be treated can be assigned a tumor gradeaccording to the International Consensus Panel on the Treatment ofBreast Cancer selected from the group consisting of grade 1, grade 1-2,grade 2, grade 2-3, or grade 3.

A cancer that is to be treated can be staged according to the AmericanJoint Committee on Cancer (AJCC) TNM classification system, where thetumor (T) has been assigned a stage of TX, T1, T1mic, T1a, T1b, T1c, T2,T3, T4, T4a, T4b, T4c, or T4d; and where the regional lymph nodes (N)have been assigned a stage of NX, N0, N1, N2, N2a, N2b, N3, N3a, N3b, orN3c; and where distant metastasis (M) can be assigned a stage of MX, M0,or M1. A cancer that is to be treated can be staged according to anAmerican Joint Committee on Cancer (AJCC) classification as Stage I,Stage IIA, Stage IIB, Stage IIIA, Stage IIIB, Stage IIIC, or Stage IV. Acancer that is to be treated can be assigned a grade according to anAJCC classification as Grade GX (e.g., grade cannot be assessed), Grade1, Grade 2, Grade 3 or Grade 4. A cancer that is to be treated can bestaged according to an AJCC pathologic classification (pN) of pNX, pN0,PN0 (I−), PN0 (I+), PN0 (mol−), PN0 (mol+), PN1, PN1(mi), PN1a, PN1b,PN1c, pN2, pN2a, pN2b, pN3, pN3a, pN3b, or pN3c.

A cancer that is to be treated can include a tumor that has beendetermined to be less than or equal to about 2 centimeters in diameter.A cancer that is to be treated can include a tumor that has beendetermined to be from about 2 to about 5 centimeters in diameter. Acancer that is to be treated can include a tumor that has beendetermined to be greater than or equal to about 3 centimeters indiameter. A cancer that is to be treated can include a tumor that hasbeen determined to be greater than 5 centimeters in diameter. A cancerthat is to be treated can be classified by microscopic appearance aswell differentiated, moderately differentiated, poorly differentiated,or undifferentiated. A cancer that is to be treated can be classified bymicroscopic appearance with respect to mitosis count (e.g., amount ofcell division) or nuclear pleomorphism (e.g., change in cells). A cancerthat is to be treated can be classified by microscopic appearance asbeing associated with areas of necrosis (e.g., areas of dying ordegenerating cells). A cancer that is to be treated can be classified ashaving an abnormal karyotype, having an abnormal number of chromosomes,or having one or more chromosomes that are abnormal in appearance. Acancer that is to be treated can be classified as being aneuploid,triploid, tetraploid, or as having an altered ploidy. A cancer that isto be treated can be classified as having a chromosomal translocation,or a deletion or duplication of an entire chromosome, or a region ofdeletion, duplication or amplification of a portion of a chromosome.

A cancer that is to be treated can be evaluated by DNA cytometry, flowcytometry, or image cytometry. A cancer that is to be treated can betyped as having 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of cellsin the synthesis stage of cell division (e.g., in S phase of celldivision). A cancer that is to be treated can be typed as having a lowS-phase fraction or a high S-phase fraction.

Any other disease in which epigenetic methylation, which is mediated byEZH2, plays a role may be treatable or preventable using compositionsand methods described herein.

Treating cancer can result in a reduction in size of a tumor. Areduction in size of a tumor may also be referred to as “tumorregression”. Preferably, after treatment with the strategies, treatmentmodalities, methods, combinations, and compositions provided herein,tumor size is reduced by 5% or greater relative to its size prior totreatment; more preferably, tumor size is reduced by 10% or greater;more preferably, reduced by 20% or greater; more preferably, reduced by30% or greater; more preferably, reduced by 40% or greater; even morepreferably, reduced by 50% or greater; and most preferably, reduced bygreater than 75% or greater. Size of a tumor may be measured by anyreproducible means of measurement. The size of a tumor may be measuredas a diameter of the tumor.

Treating cancer can result in a reduction in tumor volume. Preferably,after treatment with the strategies, treatment modalities, methods,combinations, and compositions provided herein, tumor volume is reducedby 5% or greater relative to its size prior to treatment; morepreferably, tumor volume is reduced by 10% or greater; more preferably,reduced by 20% or greater; more preferably, reduced by 30% or greater;more preferably, reduced by 40% or greater; even more preferably,reduced by 50% or greater; and most preferably, reduced by greater than75% or greater. Tumor volume may be measured by any reproducible meansof measurement.

In some embodiments, treating cancer results in a decrease in the numberof tumors. Preferably, after treatment with the strategies, treatmentmodalities, methods, combinations, and compositions provided herein,tumor number is reduced by 5% or greater relative to number prior totreatment; more preferably, tumor number is reduced by 10% or greater;more preferably, reduced by 20% or greater; more preferably, reduced by30% or greater; more preferably, reduced by 40% or greater; even morepreferably, reduced by 50% or greater; and most preferably, reduced bygreater than 75%. Number of tumors may be measured by any reproduciblemeans of measurement. The number of tumors may be measured by countingtumors visible to the naked eye or at a specified magnification.Preferably, the specified magnification is 2×, 3×, 4×, 5×, 10×, or 50×.

Treating cancer can result in a decrease in number of metastatic lesionsin other tissues or organs distant from the primary tumor site.Preferably, after treatment with the strategies, treatment modalities,methods, combinations, and compositions provided herein, the number ofmetastatic lesions is reduced by 5% or greater relative to number priorto treatment; more preferably, the number of metastatic lesions isreduced by 10% or greater; more preferably, reduced by 20% or greater;more preferably, reduced by 30% or greater; more preferably, reduced by40% or greater; even more preferably, reduced by 50% or greater; andmost preferably, reduced by greater than 75%. The number of metastaticlesions may be measured by any reproducible means of measurement. Thenumber of metastatic lesions may be measured by counting metastaticlesions visible to the naked eye or at a specified magnification.Preferably, the specified magnification is 2×, 3×, 4×, 5×, 10×, or 50×.

Treating cancer can result in an increase in average survival time of apopulation of treated subjects in comparison to a population receivingcarrier alone. Preferably, after treatment with the strategies,treatment modalities, methods, combinations, and compositions providedherein, the average survival time is increased by more than 30 days;more preferably, by more than 60 days; more preferably, by more than 90days; and most preferably, by more than 120 days. An increase in averagesurvival time of a population may be measured by any reproducible means.An increase in average survival time of a population may be measured,for example, by calculating for a population the average length ofsurvival following initiation of treatment with an active compound. Anincrease in average survival time of a population may also be measured,for example, by calculating for a population the average length ofsurvival following completion of a first round of treatment with anactive compound.

Treating cancer can result in an increase in average survival time of apopulation of treated subjects in comparison to a population ofuntreated subjects. Preferably, after treatment with the strategies,treatment modalities, methods, combinations, and compositions providedherein, the average survival time is increased by more than 30 days;more preferably, by more than 60 days; more preferably, by more than 90days; and most preferably, by more than 120 days. An increase in averagesurvival time of a population may be measured by any reproducible means.An increase in average survival time of a population may be measured,for example, by calculating for a population the average length ofsurvival following initiation of treatment with an active compound. Anincrease in average survival time of a population may also be measured,for example, by calculating for a population the average length ofsurvival following completion of a first round of treatment with anactive compound.

Treating cancer can result in increase in average survival time of apopulation of treated subjects in comparison to a population receivingmonotherapy with a drug that is not a compound of the disclosure, or apharmaceutically acceptable salt, solvate, analog or derivative thereof.Preferably, after treatment with the strategies, treatment modalities,methods, combinations, and compositions provided herein, the averagesurvival time is increased by more than 30 days; more preferably, bymore than 60 days; more preferably, by more than 90 days; and mostpreferably, by more than 120 days. An increase in average survival timeof a population may be measured by any reproducible means. An increasein average survival time of a population may be measured, for example,by calculating for a population the average length of survival followinginitiation of treatment with an active compound. An increase in averagesurvival time of a population may also be measured, for example, bycalculating for a population the average length of survival followingcompletion of a first round of treatment with an active compound.

Treating cancer can result in a decrease in the mortality rate of apopulation of treated subjects in comparison to a population receivingcarrier alone. Treating cancer can result in a decrease in the mortalityrate of a population of treated subjects in comparison to an untreatedpopulation. Treating cancer can result in a decrease in the mortalityrate of a population of treated subjects in comparison to a populationreceiving monotherapy with a drug that is not a compound of thedisclosure, or a pharmaceutically acceptable salt, solvate, analog orderivative thereof. Preferably, after treatment with the strategies,treatment modalities, methods, combinations, and compositions providedherein, the mortality rate is decreased by more than 2%; morepreferably, by more than 5%; more preferably, by more than 10%; and mostpreferably, by more than 25%. A decrease in the mortality rate of apopulation of treated subjects may be measured by any reproduciblemeans. A decrease in the mortality rate of a population may be measured,for example, by calculating for a population the average number ofdisease-related deaths per unit time following initiation of treatmentwith an active compound. A decrease in the mortality rate of apopulation may also be measured, for example, by calculating for apopulation the average number of disease-related deaths per unit timefollowing completion of a first round of treatment with an activecompound.

Treating cancer can result in a decrease in tumor growth rate.Preferably, after treatment with the strategies, treatment modalities,methods, combinations, and compositions provided herein, aftertreatment, tumor growth rate is reduced by at least 5% relative tonumber prior to treatment; more preferably, tumor growth rate is reducedby at least 10%; more preferably, reduced by at least 20%; morepreferably, reduced by at least 30%; more preferably, reduced by atleast 40%; more preferably, reduced by at least 50%; even morepreferably, reduced by at least 50%; and most preferably, reduced by atleast 75%. Tumor growth rate may be measured by any reproducible meansof measurement. Tumor growth rate can be measured according to a changein tumor diameter per unit time.

Treating cancer can result in a decrease in tumor regrowth. Preferably,after treatment with the strategies, treatment modalities, methods,combinations, and compositions provided herein, after treatment, tumorregrowth is less than 5%; more preferably, tumor regrowth is less than10%; more preferably, less than 20%; more preferably, less than 30%;more preferably, less than 40%; more preferably, less than 50%; evenmore preferably, less than 50%; and most preferably, less than 75%.Tumor regrowth may be measured by any reproducible means of measurement.Tumor regrowth is measured, for example, by measuring an increase in thediameter of a tumor after a prior tumor shrinkage that followedtreatment. A decrease in tumor regrowth is indicated by failure oftumors to reoccur after treatment has stopped.

Treating or preventing a cell proliferative disorder can result in areduction in the rate of cellular proliferation. Preferably, aftertreatment with the strategies, treatment modalities, methods,combinations, and compositions provided herein, after treatment, therate of cellular proliferation is reduced by at least 5%; morepreferably, by at least 10%; more preferably, by at least 20%; morepreferably, by at least 30%; more preferably, by at least 40%; morepreferably, by at least 50%; even more preferably, by at least 50%; andmost preferably, by at least 75%. The rate of cellular proliferation maybe measured by any reproducible means of measurement. The rate ofcellular proliferation is measured, for example, by measuring the numberof dividing cells in a tissue sample per unit time.

Treating or preventing a cell proliferative disorder can result in areduction in the proportion of proliferating cells. Preferably, aftertreatment with the strategies, treatment modalities, methods,combinations, and compositions provided herein, after treatment, theproportion of proliferating cells is reduced by at least 5%; morepreferably, by at least 10%; more preferably, by at least 20%; morepreferably, by at least 30%; more preferably, by at least 40%; morepreferably, by at least 50%; even more preferably, by at least 50%; andmost preferably, by at least 75%. The proportion of proliferating cellsmay be measured by any reproducible means of measurement. Preferably,the proportion of proliferating cells is measured, for example, byquantifying the number of dividing cells relative to the number ofnondividing cells in a tissue sample. The proportion of proliferatingcells can be equivalent to the mitotic index.

Treating or preventing a cell proliferative disorder can result in adecrease in size of an area or zone of cellular proliferation.Preferably, after treatment with the strategies, treatment modalities,methods, combinations, and compositions provided herein, aftertreatment, size of an area or zone of cellular proliferation is reducedby at least 5% relative to its size prior to treatment; more preferably,reduced by at least 10%; more preferably, reduced by at least 20%; morepreferably, reduced by at least 30%; more preferably, reduced by atleast 40%; more preferably, reduced by at least 50%; even morepreferably, reduced by at least 50%; and most preferably, reduced by atleast 75%. Size of an area or zone of cellular proliferation may bemeasured by any reproducible means of measurement. The size of an areaor zone of cellular proliferation may be measured as a diameter or widthof an area or zone of cellular proliferation.

Treating or preventing a cell proliferative disorder can result in adecrease in the number or proportion of cells having an abnormalappearance or morphology. Preferably, after treatment with thestrategies, treatment modalities, methods, combinations, andcompositions provided herein, after treatment, the number of cellshaving an abnormal morphology is reduced by at least 5% relative to itssize prior to treatment; more preferably, reduced by at least 10%; morepreferably, reduced by at least 20%; more preferably, reduced by atleast 30%; more preferably, reduced by at least 40%; more preferably,reduced by at least 50%; even more preferably, reduced by at least 50%;and most preferably, reduced by at least 75%. An abnormal cellularappearance or morphology may be measured by any reproducible means ofmeasurement. An abnormal cellular morphology can be measured bymicroscopy, e.g., using an inverted tissue culture microscope. Anabnormal cellular morphology can take the form of nuclear pleomorphism.

As used herein, the term “selectively” means tending to occur at ahigher frequency in one population than in another population. Thecompared populations can be cell populations. Preferably, a compound ofthe disclosure, or a pharmaceutically acceptable salt or solvatethereof, acts selectively on a cancer or precancerous cell but not on anormal cell. Preferably, a compound of the disclosure, or apharmaceutically acceptable salt or solvate thereof, acts selectively tomodulate one molecular target (e.g., a target protein methyltransferase)but does not significantly modulate another molecular target (e.g., anon-target protein methyltransferase). The disclosure also provides amethod for selectively inhibiting the activity of an enzyme, such as aprotein methyltransferase. Preferably, an event occurs selectively inpopulation A relative to population B if it occurs greater than twotimes more frequently in population A as compared to population B. Anevent occurs selectively if it occurs greater than five times morefrequently in population A. An event occurs selectively if it occursgreater than ten times more frequently in population A; more preferably,greater than fifty times; even more preferably, greater than 100 times;and most preferably, greater than 1000 times more frequently inpopulation A as compared to population B. For example, cell death wouldbe said to occur selectively in cancer cells if it occurred greater thantwice as frequently in cancer cells as compared to normal cells.

A composition of the disclosure, e.g., a composition comprising an EZH2inhibitor of the disclosure can modulate the activity of a moleculartarget (e.g., a target protein methyltransferase). A composition of thedisclosure, e.g., a composition comprising an immune checkpointinhibitor of the disclosure can modulate the activity of a moleculartarget (e.g., a checkpoint protein in an immune cell or progenitorthereof). Modulating refers to stimulating or inhibiting an activity ofa molecular target. Preferably, a compound of the disclosure, or apharmaceutically acceptable salt or solvate thereof, modulates theactivity of a molecular target if it stimulates or inhibits the activityof the molecular target by at least 2-fold relative to the activity ofthe molecular target under the same conditions but lacking only thepresence of said compound. More preferably, a compound of thedisclosure, or a pharmaceutically acceptable salt or solvate thereof,modulates the activity of a molecular target if it stimulates orinhibits the activity of the molecular target by at least 5-fold, atleast 10-fold, at least 20-fold, at least 50-fold, at least 100-foldrelative to the activity of the molecular target under the sameconditions but lacking only the presence of said compound. The activityof a molecular target may be measured by any reproducible means. Theactivity of a molecular target may be measured in vitro or in vivo. Forexample, the activity of a molecular target may be measured in vitro byan enzymatic activity assay or a DNA binding assay, or the activity of amolecular target may be measured in vivo by assaying for expression of areporter gene.

A composition of the disclosure does not significantly modulate theactivity of a molecular target if the addition of the compound does notstimulate or inhibit the activity of the molecular target by greaterthan 10% relative to the activity of the molecular target under the sameconditions but lacking only the presence of said compound.

As used herein, the term “isozyme selective” means preferentialinhibition or stimulation of a first isoform of an enzyme in comparisonto a second isoform of an enzyme (e.g., preferential inhibition orstimulation of a protein methyltransferase isozyme alpha in comparisonto a protein methyltransferase isozyme beta). Preferably, a compound ofthe disclosure, or a pharmaceutically acceptable salt or solvatethereof, demonstrates a minimum of a fourfold differential, preferably atenfold differential, more preferably a fifty fold differential, in thedosage required to achieve a biological effect. Preferably, a compoundof the disclosure, or a pharmaceutically acceptable salt or solvatethereof, demonstrates this differential across the range of inhibition,and the differential is exemplified at the IC₅₀, i.e., a 50% inhibition,for a molecular target of interest.

Administering a compound of the disclosure, e.g., a compositioncomprising an EZH2 inhibitor, and one or more other therapeutic agents,to a cell or a subject in need thereof results in modulation (i.e.,stimulation or inhibition) of an activity of an intracellular target(e.g., substrate). Several intracellular targets can be modulated withthe compounds of the disclosure, including, but not limited to, proteinmethyltransferase.

Treating cancer or a cell proliferative disorder can result in celldeath, and preferably, cell death results in a decrease of at least 10%in number of cells in a population. More preferably, cell death means adecrease of at least 20%; more preferably, a decrease of at least 30%;more preferably, a decrease of at least 40%; more preferably, a decreaseof at least 50%; most preferably, a decrease of at least 75%. Number ofcells in a population may be measured by any reproducible means. Anumber of cells in a population can be measured by fluorescenceactivated cell sorting (FACS), immunofluorescence microscopy and lightmicroscopy. Methods of measuring cell death are as shown in Li et al.,Proc Natl Acad Sci US A. 100(5): 2674-8, 2003. In an aspect, cell deathoccurs by apoptosis.

Preferably, the strategies, treatment modalities, methods, combinations,and compositions provided herein result in no significantly cytotoxicityto normal cells. A therapeutically effective amount of an EZH2 inhibitorand/or an immune checkpoint inhibitor is not significantly cytotoxic tonormal cells if administration of the compound in a therapeuticallyeffective amount does not induce cell death in greater than 10% ofnormal cells. A therapeutically effective amount of an EZH2 inhibitorand/or an immune checkpoint inhibitor does not significantly affect theviability of normal cells if administration in a therapeuticallyeffective amount does not induce cell death in greater than 10% ofnormal cells. In some embodiments, cell death occurs by apoptosis.

Contacting a cell with an EZH2 inhibitor and/or an immune checkpointinhibitor, can induce or activate cell death selectively in cancercells. Administering to a subject in need thereof an EZH2 inhibitorand/or an immune checkpoint inhibitor, can induce or activate cell deathselectively in cancer cells. Contacting a cell with an EZH2 inhibitorand/or an immune checkpoint inhibitor, can induce cell death selectivelyin one or more cells affected by a cell proliferative disorder.Preferably, administering to a subject in need thereof an EZH2 inhibitorand/or an immune checkpoint inhibitor, induces cell death selectively inone or more cells affected by a cell proliferative disorder.

In some aspects, the disclosure relates to a method of treating orpreventing cancer by administering an EZH2 inhibitor and/or an immunecheckpoint inhibitor, to a subject in need thereof, where theadministering results in one or more of the following: prevention ofcancer cell proliferation by accumulation of cells in one or more phasesof the cell cycle (e.g. G1, G1/S, G2/M), or induction of cellsenescence, or promotion of tumor cell differentiation; promotion ofcell death in cancer cells via cytotoxicity, necrosis or apoptosis,without a significant amount of cell death in normal cells, antitumoractivity in animals with a therapeutic index of at least 2. As usedherein, “therapeutic index” is the maximum tolerated dose divided by theefficacious dose.

One skilled in the art may refer to general reference texts for detaileddescriptions of known techniques discussed herein or equivalenttechniques. These texts include Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Inc. (2005); Sambrook et al.,Molecular Cloning, A Laboratory Manual (3^(rd) edition), Cold SpringHarbor Press, Cold Spring Harbor, N.Y. (2000); Coligan et al., CurrentProtocols in Immunology, John Wiley & Sons, N.Y.; Enna et al., CurrentProtocols in Pharmacology, John Wiley & Sons, N.Y.; Fingl et al., ThePharmacological Basis of Therapeutics (1975), Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 18^(th) edition (1990).These texts can, of course, also be referred to in making or using anaspect of the disclosure.

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any is pertinent prior art, nor does it constituteany admission as to the contents or date of the same. The inventionhaving now been described by way of written description, those of skillin the art will recognize that the invention can be practiced in avariety of embodiments and that the foregoing description and examplesbelow are for purposes of illustration and not limitation of the claimsthat follow.

The disclosure can be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. The scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

EXAMPLES Example 1: Upregulation of PD-L1 after EZH2 Inhibition

Tissue samples from the left upper arm of a subject diagnosed withepithelioid sarcoma were collected before administration oftazemetostat, and after 25 weeks of treatment. Tazemetostat wasadministered orally at 1600 mg in twice daily doses of 800 mg each.Samples were stained for PD-L1 (FIGS. 1A and 1B), CD4 (FIGS. 2A and 2B),and CD8 (FIGS. 3A and 3B). The data show that some tumors exhibitupregulated PD-L1 after treatment with tazemetostat and that such tumorsare positive for T-cell markers CD4 and CD8. Specifically, CD4+ cellswere found to make up about 2% of overall cellularity in both the pre-and post-dose tissue samples. CD4+ histiocytoid cells decreased fromabout 20% cellularity of the pre-dose sample to about 5%, upon treatmentwith tazemetostat. CD8+ immune cells increased from about 5% of overallcellularity of the pre-dose sample to about 30% of overall cellularityof the post-dose sample.

In another experiment, samples from a subject diagnosed with renalmedullary carcinoma were collected before administration oftazemetostat, and after 8 weeks of treatment. Tazemetostat wasadministered orally at 1600 mg in twice daily doses of 800 mg each.Samples were stained for PD-L1 (FIGS. 4A and 4B) and CD8 (FIGS. 5A and5B).

The data indicate that these tumors are primed for an immune responsemediated by T-cells present in the tumor, but that the immune responseis likely suppressed by the increased expression of PD-L1 in the tumor.This suggests that such tumors are treatable by a combination of EZH2inhibition and PD-1/PD-L1 inhibition. Without wishing to be bound bytheory, such treatment releases the suppression of T-cells in the tumorand unleashes an immune response upon the tumor tissue.

In a phase 1 clinical study, tazemetostat showed antitumor activity inpatients with refractory B-cell non-Hodgkin lymphoma and advanced solidtumors, including epithelioid sarcoma. Histological assessment of anon-treatment tumor specimen indicated the presence of a strong immuneinfiltrate that was neither present at baseline nor in a later specimencollected at disease progression. Moreover, modest expression of PD-L1on immune cells was observed in a tumor sample obtained after 4 weeks oftazemetostat treatment. (See Italiano et al. (2018) The Lancet Oncology19(5) 649-659; incorporated herein by reference in its entirety).

Example 2: Combination Treatment

A patient diagnosed with soft tissue sarcoma and treated withtazemetostat is biopsied after 20 weeks of treatment. Based on the tumorshowing expression of PD-L1 in more than 50% of the cells present in thetumor in the post-treatment assessment, the patient is treated with acombination of tazemetostat at an oral dose of 800 mg twice per day andatezolizumab (TECENTRIQ™) at a dose of 1200 mg as an intravenousinfusion over 60 minutes every 3 weeks until the tumor recedes.

A second patient diagnosed with soft tissue sarcoma and treated withtazemetostat is biopsied after 25 weeks of treatment. Based on the tumorshowing expression of PD-L1 in more than 80% of the cells present in thetumor and the presence of both CD4+ and CD8+ cells in the post-treatmentbiopsy, the patient is treated with a combination of tazemetostat at anoral dose of 800 mg twice per day and pembrolizumab (KEYTRUDA™) at adose of 2 mg/kg as an intravenous infusion over 30 minutes every 3weeks.

1. A method for treating a subject having a cancer that is positive fora T-cell marker comprising: (a) identifying a subject for treatmentbased on: (i) detecting a reference level of expression of PD-L1 in asubject having cancer, wherein the subject has not been administered anenhancer of zeste homolog 2 (EZH2) inhibitor, or a pharmaceuticallyacceptable salt thereof, then (ii) detecting a level of expression ofPD-L1 in the subject after the subject has been administeredtazemetostat or the pharmaceutically acceptable salt thereof, andcomparing the level of expression to the reference level of expression;and (b) administering to the subject having a cancer, wherein the PD-L1expression is increased after administration of tazemetostat or thepharmaceutically acceptable salt thereof: (i)

or the pharmaceutically acceptable salt thereof; and (ii) a PD-1inhibitor; and/or (iii) a PD-L1 inhibitor, wherein the PD-1 inhibitor isselected from Nivolumab, Pembrolizumab, Atezolizumab, Durvalumab,Avelumab, BMS-936559, AMP-224, MEDI-0680, TSR-042, BGB-108, STI-1014,KY-1003, ALN-PDL, BGB-A317, KD-033, REGN-2810, PDR-001, SHR-1210,MGD-013, PF-06801591, CX-072, or a combination thereof, and wherein thePD-L1 inhibitor is selected from Nivolumab, Pembrolizumab, Atezolizumab,Durvalumab, Avelumab, BMS-936559, AMP-224, MEDI-0680, TSR-042, BGB-108,STI-1014, KY-1003, ALN-PDL, BGB-A317, KD-033, REGN-2810, PDR-001,SHR-1210, MGD-013, PF-06801591, CX-072, or a combination thereof.
 2. Themethod of claim 1, further comprising detecting a T-cell marker in thecancer of the subject.
 3. The method of claim 1, wherein the cancer ispositive for a T-cell marker after administration of the EZH2 inhibitor.4. The method of claim 2, wherein the T-cell marker comprises CD4. 5.The method of claim 2, wherein the T-cell marker comprises CD8.
 6. Themethod of claim 1, wherein the PD-1 inhibitor and/or the PD-L1 inhibitoris selected from Nivolumab, Pembrolizumab, Atezolizumab, and Durvalumab.7. The method of claim 1, wherein the cancer is bladder cancer ortransitional cell cancer.
 8. The method of claim 1, wherein the canceris head and neck cancer or squamous neck cancer.
 9. The method of claim1, wherein the cancer is squamous cell carcinoma.
 10. The method ofclaim 1, wherein the cancer is a solid tumor.
 11. The method of claim 1,wherein the cancer is a soft tissue sarcoma.
 12. The method of claim 1,wherein the cancer is colorectal cancer or pancreatic cancer.
 13. Themethod of claim 12, wherein the pancreatic cancer is selected fromductal adenocarcinoma, adenosquamous carcinoma, pleomorphic giant cellcarcinoma, mucinous adenocarcinoma, osteoclast-like giant cellcarcinoma, and mucinous cystadenocarcinoma.
 14. The method of claim 1,wherein the cancer is breast cancer.
 15. The method of claim 1, whereinthe cancer is lung cancer.